WO2020062977A1 - Gene-encoding artificial photosynthesis protein and application thereof - Google Patents

Abstract

Provided are a gene-encoding artificial photosynthesis protein and an application thereof. Specifically, provided is an artificial photosynthesis protein, the protein comprising a fluorescent protein (FP) in which a chromophore amino acid residue is modified by a photosensitizer so as to be converted into a photosensitized protein (PSP). Provided is a photosensitive carbon dioxide reductase, comprising the artificial photosynthesis protein and a carbon dioxide reduction catalyst conjugated to the artificial photosynthesis protein. The constructed artificial photosynthesis protein and photo-sensitive carbon dioxide reductase are more compatible with a wide range of biological systems, do not rely on precious metals, may potentially sensitize a plurality of challenging chemical transformations, and relate to diverse fields such as solar energy conversion, photobiology, environmental restoration and industrial biology.

Description

Gene-encoded artificial photosynthesis protein and application thereof Technical field

The invention provides an artificial photosynthesis protein that can be genetically encoded and its application. Specifically, the genetically-encoded artificial photosynthetic protein provided by the present invention can simulate the absorption of light energy by a natural photosynthesis system and catalyze the reduction of carbon dioxide to carbon monoxide.

Background technique

In recent years, the development of efficient carbon dioxide fixation methods to cope with the increasing atmospheric carbon dioxide concentration has become a key issue in the field of chemical research. Among them, the plant photosynthesis system as a natural solution has attracted widespread attention due to its advantages such as cleanliness, self-assembly, and efficient photo-charge separation efficiency. However, the carbon dioxide reduction efficiency of natural photosynthesis systems is relatively low compared to chemical small molecule catalysts. In addition, the natural photosynthesis system consists of complex membrane protein subunits and multiple coenzymes, which brings inconvenience to research and practical applications.

Photosynthesis is the most important process on earth, which converts solar energy into chemical energy and converts carbon dioxide (CO ₂ ) into biomass ^1-4 . At present, researchers have great interest in how to improve the efficiency of photosynthesis and reuse light systems to promote challenging chemical transformations. However, there are significant technical challenges ^5-12 . First, because the natural photosynthetic system is composed of a large number of membrane proteins, enzymes and cofactors, it is very difficult to genetically modify the photosynthesis mechanism. Second, although photosystem I and photosystem II can work together to reduce NADP ⁺ to NADPH (E ⁰ = -320mV, relative to standard hydrogen electrode (SHE), all reduction potentials are relative to SHE); however, NADPH cannot promote carbon dioxide (CO ₂ ) to carbon monoxide ( CO) direct reduction (E ⁰ = -520 mV).

Summary of the Invention

Brief description of the invention

In order to solve these problems, the applicant has been committed to the application of synthetic biological methods to develop artificial photosynthesis systems that can be genetically coded, so that they have the advantages of both natural light systems and chemical small molecule catalysts. This artificially designed photosynthetic protein can not only provide new ideas for the study of carbon dioxide reduction methods, but also provide the basis for the evolution of artificial living organisms with non-natural photocatalytic activity.

In some embodiments, the present invention relates to one or more of the following:

What is claimed is: 1. An artificial photosynthesis protein comprising a fluorescent protein (FP) in which a chromophore amino acid residue is modified by a photosensitizer to be converted into a photosensitized protein (PSP).

2. The artificial photosynthetic protein according to item 1, wherein the fluorescent protein includes a dark blue fluorescent protein such as Sirius, a blue fluorescent protein such as EBFP, Azurite, EBFP2, TagBFP, a cyan fluorescent protein such as ECFP, Cerulean, CyPet, mTurquoise, mTFP1 (Teal), Midoriishi-Cyan, green fluorescent proteins such as GFP, UKG, EGFP, Emerald, Superfolder, yellow fluorescent proteins such as YFP, sfYFP, EYFP, Venus, Citrine, YPet, PhiYFP, orange fluorescent proteins such as mHoneydew, mBanana, mKO , MKOκ, mOrange, mOrange2, red fluorescent proteins such as TagRFP, TagRFP, 158T, mRuby, mCherry, deep red fluorescent proteins such as Katushka, mKate, mKate2, mPlum, E2-Crimson, mNeptune, Tag657, eqFP650, eqFP670, near-infrared fluorescent proteins such as mIFP, a bacterial photochrome protein such as BphP, a phycobiliprotein such as phycoerythrin CPE, phycocyanin PEC, phycocyanin CPC, phycocyanin APC, or a variant thereof.

3. The artificial photosynthetic protein according to item 1 or 2, wherein the photosensitizer is a photosensitizer encoded by an extended genetic code, such as benzophenone-alanine BpA.

4. The artificial photosynthetic protein according to any one of items 1-3, wherein the chromophore amino acid residues include, for example, amino acid residues corresponding to positions 65, 66, and 67 of the green fluorescent protein GFP, and particularly correspond to Tyrosine residue at position 66 of the green fluorescent protein GFP.

5. The artificial photosynthetic protein according to any one of items 1-4, wherein the modification includes introducing the photosensitizer through amino acid residue insertion, substitution, deletion to change the amino acid sequence encoding a wild-type fluorescent protein, such as extended genetics Coded photosensitizers, such as benzophenone-alanine BpA.

6. The artificial photosynthetic protein according to any one of items 1-5, further comprising modification of amino acid residues other than chromophore residues, such as amino acid residue insertions, substitutions, and deletions.

7. The artificial photosynthetic protein according to any one of items 1-6, wherein the modification of the amino acid residue includes a modification capable of obtaining one or more of the following properties: 1) adjusting the photochemical characteristics of the chromophore, such as making it The photoexcited state has sufficient oxidizing ability, which can oxidize the weak sacrificial reducing agent to produce a strong reducing group that promotes the reduction of the carbon dioxide reduction catalyst; 2) adjust the distance between the chromophore and the catalytic center to promote the transition from the chromophore to the catalyst The continuous electron transfer step of the center prevents unfavorable charge recombination; and / or 3) adjusts the microenvironment of the catalytic center to optimize the transfer of protons and electrons.

8. The artificial photosynthetic protein according to any one of items 1 to 7, which is an artificial photosynthetic protein having one or more of the following properties: (1) can effectively absorb visible light; (2) when absorbing photons, Able to transform the long-existing photoexcited state (PSP *), thereby promoting the electron transfer reaction, leading to the formation of PSP radicals (PSP ·); and / or (3) PSP · is a strong reducing agent that can drive the carbon dioxide reduction catalyst. reduction.

9. The artificial photosynthetic protein according to any one of items 1-8, comprising

a) the amino acid sequence shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,

b) has a sequence shown in any of the above with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher Amino acid sequence of amino acid identity;

c) has one or more sequences (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16 17, 18, 19, 20, 21, 22, 23, 24, 25 or more, preferably one or several, such as 1, 2, 3, 4, 5 or more) amino acid sequences with amino acid differences.

10. A photosensitive carbon dioxide reductase, comprising the artificial photosynthetic protein according to any one of items 1-9 and a carbon dioxide reduction catalyst conjugated to the artificial photosynthetic protein.

11. The photosensitive carbon dioxide reductase of item 10, wherein the carbon dioxide reduction catalyst comprises a terpyridine nickel (II) complex.

12. The photosensitive carbon dioxide reductase according to item 10 or 11, wherein the carbon dioxide reduction catalyst is directly conjugated to an amino acid residue of the artificial photosynthesis protein, or indirectly conjugated to the artificial photosynthesis protein through a linker. Amino acid residues.

13. The photosensitive carbon dioxide reductase of item 12, wherein the amino acid residues conjugated to the carbon dioxide reduction catalyst include amino acid residues introduced by amino acid residue modification.

14. The photosensitive carbon dioxide reductase according to any one of items 10-13, wherein the carbon dioxide reduction catalyst is directly or indirectly conjugated to a cysteine residue, such as an cysteine residue introduced by modification of an amino acid residue .

15. The photosensitive carbon dioxide reductase according to any one of items 10 to 14, which is free of precious metals and / or independent of precious metals.

16. An artificial photosynthesis system comprising the artificial photosynthesis protein according to any one of items 1-9 or the photosensitive carbon dioxide reductase according to any one of items 10-15, preferably the artificial photosynthesis system is Gene-coded artificial photosynthesis system.

17. A method for preparing an artificial photosynthetic protein, comprising: modifying a chromophore amino acid residue of a fluorescent protein (FP) with a photosensitizer, thereby converting it into a photosensitized protein (PSP).

18. The method of item 17, comprising the further modification of amino acid residues other than chromophore residues, such as amino acid residue insertions, substitutions, and deletions, for example, by substitution of charged residues to regulate the PSP chromophore Reduction potential.

19. The method of item 17 or 18, which comprises detecting a color change of the modified product to determine the occurrence of a photochemical reaction.

20. The method of any of items 17-19, comprising detecting the reducing ability of the modified product, such as a photochemical reduction of a weak reducing agent such as ascorbic acid.

21. The method of any of items 17-19, comprising 1) performing UV-Vis titration, such as UV-Vis titration at different pH conditions; 2) collecting X-band electron spin resonance data; 3) Perform electrochemical detection of the protein; 4) detect the reactivity with oxygen; and / or 5) determine the crystal structure, such as by X-ray diffraction.

22. A fusion protein comprising the artificial photosynthesis protein according to any one of 1-9.

23. A nucleic acid molecule encoding the artificial photosynthesis protein according to any one of items 1-9 or the fusion protein according to item 22, preferably the codons of the nucleic acid molecule are optimized to fit in the corresponding host cell Expression, for example, the codons of the nucleic acid molecule are optimized for expression in mammalian cells.

24. A recombinant expression system comprising the nucleic acid molecule according to item 23.

25. A recombinant host cell comprising the expression system of item 24.

In some embodiments, the photosensitizer is able to use light energy to turn a weak reducing agent into a strong reducing agent, so it is a key component in natural and artificial photosynthesis mechanisms. In this application, the inventors started with the modification of light-sensitive proteins, overcame various restrictions in the prior art, and reasonably designed a genetically-encoded artificial photosensitizers protein (PSP) and a light-sensitive CO ₂ reductase. The light-sensitive CO ₂ reductase mutates a specific site (for example, position 95) of PSP to cysteine, and then specifically conjugates a terpyridine nickel (II) complex (for example, N- ( 2,6,2-terpyridin-4-yl) -iodoacetamide is modified by adding nickel ion).

By expanding the genetic code, the inventor incorporated benzophenone-alanine (BpA) at the 66th position of superfolder yellow fluorescent protein (sfYFP, amino acid sequence is shown in SEQ ID NO: 1), After further expansion of the genetic code, a series of genetically encoded artificial light-sensitive proteins (PSPs) were obtained.

On the basis of PSP, the inventors further introduced mutations in other specific amino acid residues, thereby specifically conjugating the terpyridine nickel (II) complex on the PSP to obtain a protein conjugate having the activity of photocatalytic carbon dioxide to carbon monoxide , Which is called photocatalytic carbon dioxide reductase (PSP2 95C terpyridine Ni (II), referred to as PSP2T for short). The molecular weight of PSP2T is only 27 kD, which can be prepared by chemically modifying the genetically expressed PSP protein with a terpyridine nickel (II) complex in vitro. PSP2T works in water / DMF solution and does not require precious metals. The conversion quantum efficiency of CO ₂ / CO is 2.6%, which is much higher than most photocatalysts using nanocrystals or small molecule photosensitizers under similar conditions. The CO ₂ / CO conversion has a quantum efficiency of ³ . In addition to the simplicity of PSP2T, the inventors were inspired in the study of PSP2T and understood the essence of complex photosynthesis mechanisms: visible light absorption, generation of strong reducing power, and carbon dioxide reduction.

During light absorption, PSP is effectively converted into a long-lived triplet excited state PSP * (long-lived triplet excited state PSP *), and further electron transfer causes weak reducing power (such as NADH with weak reducing power) As a result, the super reducing agent PSP · (E ⁰ <-1.14V) with greatly enhanced reducing power is produced. Experiments have shown that EuDTPA (Europium (II) Diethylenetriaminepentaacetic acid, diethylenetriaminopenta (II)) cannot reduce PSP to a free radical state without photoexcitation. EuDTPA has a very low reduction potential and its standard reduction potential. It is E ⁰ = -1.14V.

Next, the inventors analyzed the crystal structure of PSP ·, which provides the necessary atomic structure information for the use of PSP · to promote a new enzyme reaction. Importantly, the inventors have demonstrated through research that three variables important for PSP2T activity can be conveniently and independently optimized by mutagenesis, resulting in significantly improved carbon dioxide reduction activity. First, the photochemical properties of the chromophore can be fine-tuned to make its photo-excited state sufficiently oxidizing, capable of oxidizing weak sacrificial reducing agents (SR), thereby generating strong reducing groups that can be used to promote the reduction of carbon dioxide reduction catalysts. Second, it is possible to fine-tune the distance between the chromophore and the catalytic center, thereby promoting a continuous electron transfer step from the chromophore to the catalytic center, and at the same time preventing adverse charge recombination steps; third Since electrons and protons are required for carbon dioxide reduction, the microenvironment of the catalytic center can be fine-tuned to optimize the transfer of protons and electrons.

However, it is difficult to achieve easy and independent optimization of the above three variables by modifying natural photosynthesis systems, nanocrystals or small molecule photosensitizers. Therefore, the inventors' work represents a promising new approach to photoenzyme design, which can provide important models for studying multiple electron / proton transfers on proteins, and can be used in renewable energy, carbon dioxide utilization, reduction of greenhouse gas emissions, and photooxidation. It is also widely used in catalysts.

Detailed description of the invention

As known to those skilled in the art, fluorescent proteins include proteins that emit light upon encountering excitation light. In the present invention, the fluorescent protein is not particularly limited. For example, the fluorescent protein may include a dark blue fluorescent protein such as Sirius, a blue fluorescent protein such as EBFP, Azurite, EBFP2, TagBFP, a cyan fluorescent protein such as ECFP, Cerulean, CyPet, mTurquoise, mTFP1 (Teal), Midoriishi-Cyan, green fluorescent proteins such as GFP, UKG, EGFP, Emerald, Superfolder, yellow fluorescent proteins such as YFP, sfYFP, EYFP, Venus, Citrine, YPet, PhiYFP, orange fluorescent proteins such as mHoneydew, mBanana, mKO , MKOκ, mOrange, mOrange2, red fluorescent proteins such as TagRFP, TagRFP, 158T, mRuby, mCherry, deep red fluorescent proteins such as Katushka, mKate, mKate2, mPlum, E2-Crimson, mNeptune, Tag657, eqFP650, eqFP670, near-infrared fluorescent proteins such as mIFP, a bacterial photochrome protein such as BphP, a phycobiliprotein such as phycoerythrin CPE, phycocyanin PEC, phycocyanin CPC, phycocyanin APC, or a variant thereof. In some embodiments, the fluorescent protein can be from any suitable source, such as corals such as Galaxea fascicularis, anemones, jellyfish, and the like. For example, green fluorescent protein GFP (GenBank Accession No. AAA27722) derived from jellyfish (Aequorea victoria) is known in the art, and mutant fluorescent proteins such as YFP, CFP, EGFP, EYFP, ECFP, and the like. The GFP gene encoding sequences registered in GenBank also include AEVGFPAM62653, AVGFP1X83959, AVGFP2X83960, and AEVGFPL29345. Appropriate modifications of fluorescent proteins other than photosensitizers can also be included in the invention. In some embodiments, the present invention may include modifications to the GFP chromophore residue tripeptide sequence at amino acids 65, 66, 67-serine, tyrosine, glycine, for example, modifications to the 65th serine residue may include Alanine, leucine, cysteine, valine, isoleucine, or threonine are substituted to obtain a protein with a red-shifted spectrum. In some embodiments, the chromophore residues of the fluorescent protein may include, for example, SHG (EBFP2), TWG (ECFP, mCerulean), AYG (mTFP1), TYG (mEGFP, mEmerald), GYG (EYFP, Mvenus, mCitrine, YPet) ), CYG (mKO), MYG (tdTomato, TagRFP, mCherry, mKate, mPlum), QYG (mRFP1), etc. In some embodiments, the invention includes, for example, amino acid residue modifications known in the art, such as modification of amino acid residue tyrosine (Y) at position 66, such as Y66H includes a blue-emitting fluorescent protein; V163A- S175G has heat resistance and enhanced fluorescence intensity; F64I, F64V, F64A, F64G, F64L have enhanced fluorescence intensity; F64L-Y66H-Y145F-L236R, F64L-Y66H-Y145F-V163A-S175G-L236R, Y66H-Y145F- V163A-S175G and F64L-Y66H-Y145F are related to photostable fluorescent proteins.

The invention also relates to a fluorescent protein with a molecular weight of only about 27 kD discovered by the inventors in a previous study, which also has the potential to be transformed into a photosynthetic protein similar to the natural light system. First, it was found that after the fluorescent protein is excited by light, its chromophore can generate an intermediate with high reducing activity, and this intermediate can efficiently transfer electrons to an electron acceptor located outside the β-fold barrel of the protein. In addition, using gene codon extension technology, the inventors can specifically insert unnatural amino acids, thereby replacing the tyrosine in the original chromophore. This allows researchers to rationally design the chemical structure of the fluorescent chromophore of a fluorescent protein and optimize its photochemical properties such as its absorption spectrum, excited state lifetime, and free radical reduction potential.

The core problem in designing efficient carbon dioxide photoreductive proteins based on fluorescent protein mutants is how to extend the life of the reducing intermediate state generated by the excitation of its chromophore and reduce its reduction potential. In the present invention, the inventors chose a tyrosine analog (BpA) with a benzophenone substituent to modify the chromophore. Benzophenone is a commonly used photosensitizer in organic photocatalysis. When it is irradiated with light of a certain wavelength, its excited state will cross between systems into a triplet state with a longer life. This triplet state in turn reacts with the sacrificial reducing agent to form a highly active free radical state, catalyzing the downstream redox reaction. After the codon extension method was used to insert the chromophore of BpA to modify the fluorescent protein, the newly generated fluorescent protein (PSP) retained this characteristic. Studies using transient absorption spectroscopy show that after being excited by light, the new chromophores inserted into BpA can be almost completely converted to the triplet state; in the presence of a sacrificial reducing agent (such as ascorbic acid), the triplet intermediate is equivalent to rapid oxidation Sacrifice the reducing agent, thereby generating a free radical state. This free radical is protected by the protein backbone, so it can exist stably for more than 10 minutes in the absence of oxygen. On the other hand, electrochemical analysis of small molecule analogues of chromophores shows that the reduced single-electron generated state has a reduction potential close to -1.5V. This not only meets the need to reduce carbon dioxide, it is also lower than known natural biological reducing agents.

After obtaining a fluorescent protein that can be excited by light to generate a strong reducing activity, the inventors further applied chemical or biological methods to introduce a terpyridine nickel complex at a specific site on the outer surface of the PSP (this is a known small molecule carbon dioxide Reduction electrochemical catalyst). This modified protein has the activity of reducing carbon dioxide to generate carbon monoxide under light conditions. Its 24-hour carbon monoxide conversion number is up to 120, and the photon quantum yield is 2.6%, which is higher than most of the reported carbon dioxide photoreduction catalysts. This illustrates the optimization of electron transfer and the improvement of activity based on the self-assembly properties of proteins.

In a first aspect, the present invention provides a genetically-encoded artificial photosynthesis protein (PSP), which is extended by a genetic codon, in a superfolder yellow fluorescent protein (sfYFP, amino acid sequence such as SEQ ID ID NO: (Shown in 1) The amino acid position 66 is obtained by mixing benzophenone-alanine (BpA). In other words, the 66th tyrosine (Tyr, Y) of sfYFP was replaced by benzophenone-alanine (BpA), and this amino acid substitution was introduced by the genetic codon expansion method. sfYFP (SEQ ID NO: 1) is an artificially synthesized protein whose amino acid sequence is 88% similar to the Mesorhizobium® loti sequence.

In one embodiment, benzophenone is incorporated at the 66th amino acid position of the superfolder yellow fluorescent protein (sfYFP, amino acid sequence shown in SEQ ID NO: 1) by genetic codon expansion- The artificial photosynthetic protein obtained from alanine (BpA) is shown in SEQ ID NO: 2 and named sfYFP-BpA66.

On the basis of sfYFP-BpA66, the tyrosine (Tyr) at 203 in sfYFP was mutated to phenylalanine (Phe) by genetic codon mutation. The double mutant sfYFP-BpA66-Phe203 was named PSP1. Its amino acid sequence is shown in SEQ ID NO: 4. Furthermore, a triple mutant sfYFP in which tyrosine (Tyr) at 203 of sfYFP was mutated to aspartic acid (Asp) and histidine (His) at 148 was mutated to glutamic acid (Glu). -BpA66-Asp203 Glu148 is called PSP2, and its amino acid sequence is shown in SEQ ID NO: 6.

Among them, PSP2 can reversibly form PSP2 radicals (PSP2 ·) in the photochemical reaction. The reversible formation of PSP2 · indicates that although PSP2 · can react with oxygen (a common characteristic of almost all super-reducing free radicals), the reaction produces PSP2 without destroying the chromophore. Since various carbon dioxide reducing agents are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of the catalyst. In addition, the decay lifetime of the PSP2 triplet excited state (PSP2 *, Fig. 4a / b) is 123 μs. The reduction potential of PSP2 · is less than -1.14V. The pKa of PSP2 · is 10.6.

On the basis of PSP2, the 95th amino acid was mutated from glutamic acid (Glu) to cysteine (Cys, single letter symbol: C) to obtain a PSP2-95Cys mutant (also denoted as PSP2-95C), which The amino acid sequence is shown in SEQ ID NO: 8. The mutant was finally conjugated in the presence of a divalent nickel ion after modification with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in Example 1). Named as PSP2T1, PSP2T1 has a higher carbon dioxide reducing activity, in which N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specifically conjugated at the 95th and a half positions of the PSP2-95C mutant On cystine residues.

In order to study whether the presence of a local proton donor can improve the catalytic efficiency, the inventors mutated valine (Val) at position 93 and threonine (Thr) at position 97 to tyrosine on the basis of PSP2T1 ( Tyr, single-letter symbol: Y), to obtain mutant PSP2-95C93Y97Y, whose amino acid sequence is shown in SEQ ID NO: 10. This mutant was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- (2,6,2-terpyridine-4) -Yl) -iodoacetamide is specifically conjugated to cysteine at position 95). The final conjugate obtained in the presence of divalent nickel ions is named PSP2T2, which shows a significantly increased number of carbon monoxide conversions ( TON) (Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).

In fact, in PSP2T1 and PSP2T2, a terpyridine nickel (II) complex is conjugated to the cysteine at position 95. Among them, the terpyridine nickel (II) complex is a known small molecule carbon dioxide reduction electrochemical catalyst.

Throughout the specification, BpA66 indicates that the 66th tyrosine of a superfolded yellow fluorescent protein (sfYFP) is mutated to BpA, or Tyr66BpA (that is, the number indicates the amino acid position of the mutation, and the left side of the number is the amino acid residue before the mutation Group, amino acid residues to the right of the number). Phe203 indicates that the tyrosine (Tyr) at position 203 of the superfolded yellow fluorescent protein (sfYFP) is mutated to phenylalanine (Phe), and can also be expressed as Tyr203Phe. 95Cys indicates that the 95th position of the superfolded yellow fluorescent protein (sfYFP) is mutated from glutamic acid (Glu) to cysteine (Cys, single letter symbol: C), and can also be expressed as Glu95Cys or 95C. Amino acid mutations at other positions are also expressed as described above.

In a second aspect, the present invention provides a photosensitive carbon dioxide reductase (PSP2 terpyridine Ni (II), also known as PSP2-terpyridine nickel complex conjugate, referred to as PSP2T), which Gene-coded artificial photosynthesis protein (PSP) specific site (for example, position 95) is mutated to cysteine, and then N- (2,6,2-terpyridine-4- ) -Iodoacetamide, and in the presence of divalent nickel ions, the divalent nickel ions and N- (2,6,2-terpyridin-4-yl) -iodoethyl conjugated to cysteine Obtained by amide coordination.

In one embodiment, the conjugation of artificial photosynthetic protein (PSP) with terpyridine is achieved by bringing the corresponding artificial photosynthetic protein to N- (2,6,2-terpyridin-4-yl) -iodoacetamide (implementation The compound 7) synthesized in Example 1 was reacted. Specifically, N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specifically conjugated to a single cysteine residue introduced in the PSP. In a preferred embodiment, cysteine is introduced at position 95 in the PSP protein, and N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specific to the cysteine Conjugated PSP-terpyridine nickel (II) complex conjugates obtained in the presence of divalent nickel ions have the activity to catalyze the photochemical reaction of carbon dioxide to form carbon monoxide.

In a preferred embodiment, the mutant PSP2-95C is modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- ( 2,6,2-terpyridin-4-yl) -iodoacetamide is conjugated to cysteine at position 95). The final conjugate obtained in the presence of divalent nickel ion is named PSP2T1, which has a relatively High carbon dioxide reducing activity.

The mutant PSP2-95C93Y97Y was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in Example 1), and PSP2T2 was obtained in the presence of divalent nickel ions. Significantly increased carbon monoxide conversion (TON) (Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).

In one embodiment, the terpyridine modification of the mutant PSP2-95C or PSP2-95C93Y97Y can be achieved by the organism. For example, transfer the PSP2-95C or PSP2-95C93Y97Y expression vector into an appropriate host cell, and add an appropriate expression inducer and terpyridine (for example, N- (2,6,2-terpyridine-4) to the medium. -Yl) -iodoacetamide), after expression, appropriate divalent nickel ions are added to obtain the corresponding protein-terpyridine nickel (II) complex conjugate.

In a third aspect, the present invention provides a method for photocatalytic reduction of carbon dioxide by using the photosensitive carbon dioxide reductase according to the second aspect of the present invention. The method includes the following steps: adding the second aspect of the present invention to the reaction system. The photo-sensitive carbon dioxide reductase (for example, PSP2T1 or PSP2T2, preferably PSP2T2) and the sacrificial reducing agent are irradiated with visible light to reduce carbon dioxide in the reaction system to carbon monoxide. Visible light irradiation can be performed using a xenon lamp that simulates the sunlight spectrum. Those skilled in the art can understand that “carbon dioxide in the reaction system” includes a case where the reaction system includes related reactants capable of generating carbon dioxide.

In some embodiments, the present invention also provides one or more of the following:

A genetically-encoded artificial photosynthesis protein obtained by incorporation of benzophenone-alanine BpA at the amino acid position 66 of the superfolded yellow fluorescent protein sfYFP through genetic codon expansion, and its amino acid sequence As shown in SEQ ID NO: 2.

2. The artificial photosynthesis protein according to item 1, further comprising a Tyr203Phe mutation, and the amino acid sequence is shown in SEQ ID NO: 4.

3. The artificial photosynthesis protein according to item 1, further comprising Tyr203Asp and His148Glu mutations, and the amino acid sequence is shown in SEQ ID NO: 6.

4. The artificial photosynthetic protein according to item 3, wherein the triplet excited state of the protein has an attenuation life of 123 μs.

5. The artificial photosynthesis protein according to item 3, further comprising a Glu95Cys mutation, and the amino acid sequence is shown in SEQ ID NO: 8.

6. The artificial photosynthesis protein according to item 3, further comprising mutations of Glu95Cys, Val93Tyr and Thr97Tyr, and the amino acid sequence is shown in SEQ ID NO: 10.

7. A photosensitive carbon dioxide reductase, which is a specific conjugate of the artificial photosynthetic protein of item 5 or 6 and a terpyridine nickel complex, wherein the conjugate is obtained by dissolving N- (2,6,2 -Terpyridin-4-yl) -iodoacetamide is specifically conjugated to the cysteine at position 95, and the divalent nickel ion is further conjugated to the cysteine in the presence of a divalent nickel ion. N- (2,6,2-terpyridin-4-yl) -iodoacetamide is obtained by coordination.

8. The photosensitive carbon dioxide reductase according to item 7, wherein the specific conjugate of the artificial photosynthetic protein of item 6 with a terpyridine nickel complex has a quantum yield of 2.6%.

The advantage of the present invention is that compared with semiconductor nanocrystals and small molecule photosensitizers, the PSP constructed by the present invention provides unique advantages, for example, it has higher compatibility with a wide range of biological systems, does not rely on precious metals, Switchable photochemical properties, and the ability to self-assemble into precise three-dimensional structures, can allow for modular expansion of its functions and accurate mechanism characterization. As a result, PSP can potentially sensitize a variety of challenging chemical transformations, involving diverse fields such as solar energy conversion, photobiology, environmental restoration, and industrial biology.

The photosynthetic protein synthesized by the present invention can be synthesized in the organism through genetic coding, can react with oxygen to form a free radical form reversibly without destroying the chromophore, and is a robust photochemical reaction catalyst. In addition, the triplet excited state produced by light irradiation has a long decay life, which is more conducive to reducing carbon dioxide.

BRIEF DESCRIPTION OF THE DRAWINGS

The above features and advantages of the present invention will be more apparent from the following detailed description in conjunction with the accompanying drawings, in which:

Figure 1. Proper design of PSP and PSP2T.

(a) Reduction potentials of protein oxidation centers and small molecules. Ni (II) (terpy): nickel-terpyridine complex. CdS: cadmium sulfide quantum dot.

(b) Above: Structures of tyrosine (left) and benzophenone-alanine (BpA, right). Bottom: In the presence of dithionite, sfYFP, PSP1 and PSP2 are irradiated with a 405nm laser pen for 2 minutes, and then they are taken with a digital camera.

(c) Ultraviolet-visible absorption (UV-Vis) spectra of PSP1, PSP1 ·, PSP2, and PSP2 · in a 100 mM Tris-HCl buffer at pH 7 using dithionite as a sacrificial reducing agent (SR). Dithionite does not absorb at wavelengths above 350 nm.

(d) UV-Vis spectra of PSP2 at different pH.

(e) X-band electron spin resonance (X-band ESR) spectra of PSP2 (basically a horizontal line) and PSP2 · (a curve with peaks and troughs) using NADH as a reducing agent before and after 405 nm laser irradiation. NADH is used as a sacrificial reducing agent because it has no background ESR signal. Insertion: Photographs of ESR tube containing PSP2 before and after 405nm laser irradiation (turned red).

(f) Small molecule analogue of chromophore in N, N-dimethylformamide (DMF) (E) -4- (4-benzoylbenzylidene) -1,2-dimethyl -1H-imidazole-5 (4H) -one (BpAChm, compound 6, see Example 1) was measured by cyclic voltammetry (CV).

Figure 2. Crystallographic characterization of PSP.

(a) Device setup for X-ray diffraction of PSP2 crystal.

(b) A photograph of a PSP2 crystal irradiated with a 405 nm laser pen in the presence of 160 mM dithionite in a crystallization buffer. After irradiation, the solution turned red. After stopping the irradiation, the solution gradually returned to its original color.

(c) Before the photochemical reduction, a snapshot of the chromophore structure in the ground state shows that the dihedral angle of the two benzene rings of the BpA66 residue is 58 °.

(d) A snapshot of the PSP2 · chromophore structure acquired under continuous 405nm laser irradiation. The dihedral angle of the two benzene rings in PSP2 · is 29.1 °.

(e) The arrow indicates the apparent rotation of the benzene ring of the BpA66 residue in PSP2 · (red) after 405 nm laser irradiation compared to the dark state (yellow). The dihedral angle of O (21) -C (7) -C (6) -C (5) (see Fig. 7) changed from -146.9 ° to -24.1 °, which resulted in a complete inversion of the carbonyl direction of BpA.

Figure 3. Design and characterization of PSP2T.

(a) Schematic of the proposed PSP2T catalytic mechanism.

(b) CO formation from N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7, see Example 1) modified by various PSP2 monocysteine variants Conversions (TON). The distance from the catalyst to the PSP2 chromophore is shown.

(c) TON formed by CO catalyzed by the PSP2T mutant.

(d) CO formation catalyzed by PSP2T1 / 2 varies with irradiation time.

The error bars in all cases are standard errors (s.d.) (n = 3).

Figure 4. Instantaneous absorption spectra (a, c) and spectral dynamics (b, d) of PSP2.

(a) Instantaneous absorption spectrum of PSP2.

(b) Kinetic trajectories of PSP2 recorded at 370 nm, 430 nm, and 570 nm.

(c) Instantaneous absorption spectrum of PSP2 in the presence of 100 mM ascorbate (Asc). Ascorbate (Asc) has no absorption at 355 nm and is stable in air, and therefore acts as a reducing agent.

(d) Kinetic trajectory recorded at 430 nm in the presence of 0, 50, 100, 150 mM ascorbate (Asc).

(e) Summary of photochemical processes of PSP. After photon absorption, the electron transitions from the ground state of S ₀ to the singlet excited state of the higher energy state of S ₁ . In this system, the S ₁ state has almost no chance to return to the S ₀ ground state through fluorescence, but instead crosses to the triplet PSP * by intersystem crossing (ISC) nearly 100%. The lifetime of this triplet PSP * is about 123 μs. If a sacrificial reducing agent (SR) is present, the triplet state can acquire an electron and become a protein free radical state PSP. In the absence of oxygen, a protein free radical state containing benzophenone-alanine (BpA) (PSP ·) has a lifetime of more than 1 s.

(f) Proposed PSP2T photocatalytic mechanism. ISC: intersystem crossing; ₁ S: PSP single excited state; T _1: PSP triplet excited state (PSP *).

Figure 5.a, BpA structure; b, BpAChm structure, BpAChm simulates the chromophore structure of PSP. c, UV-Vis spectrum of BpA before and after 405nm laser irradiation; d, UV-Vis spectrum of BpAChm before and after 405nm laser irradiation; in both cases, in the presence of 10 mM dithionite Under conditions, no benzophenone radicals were observed after irradiating the sample with a 405 nm laser pen for more than 10 minutes. e. UV-Vis spectra of PSP1 after treatment with dithionite alone (10 mM) or with 405 nm laser irradiation (10 minutes). No PSP1 spectral changes were observed.

Figure 6.a, UV-Vis spectra of PSP1 and PSP2 after 405nm laser irradiation for 10 minutes; conditions: 50 μMPSP, 100 mM Tris-HCl pH 7.0 buffer, 100 mM Asc (ascorbate); b, at 100 mM Tris-HCl pH UV-Vis spectrum of PSP2 in 7.0 buffer, irradiated with 405nm laser pen for 10 minutes in the absence of Asc, or treated with 100mMAsc in the absence of laser irradiation; in both cases, there is no PSP2 spectrum changes were observed and no PSP2 · radical formation was obtained; c, UV-Vis spectra of PSP1 · at different pH. In 100 mM buffers of different pH values: Tris-HCl (pH 6.0-8.0), Glycine-NaOH (pH 9-10), PSP1 was irradiated with a 405nm laser pen for 10 minutes to obtain PSP1 ·. d. Reduce the UV-Vis spectrum of PSP2 with Eu (II) -DTPA. No PSP2 spectrum change was observed, and no PSP2 · radical formation was obtained. Conditions: 65 μM PSP2, 100 mM Tris-HCl pH 8.0 buffer, 5 mM Eu (II) -DTPA.

Figure 7. Structure of benzophenone (BP), BP-COOH, BP neutral radical, BP anionic group, PSP1 chromophore, PSP2 chromophore, PSP2 neutral radical, PSP2 anionic group and BpAChm Naming.

Figure 8. Circular dichroism (CD) spectra of PSP2 in different pH buffers. Conditions: 10 μM PSP2 in 100 mM buffers with different pH values (that is, Tris-HCl (pH 6.0-8.0), glycine-NaOH (pH 9-10.6), Carbonate-NaOH (pH 11.4-11.8)) is placed on quartz In a cup (200 μL, 1 cm path), the spectrum was measured with a circular dichroism spectrometer at room temperature.

Figure 9. The formation of PSP2 · is reversible. Conditions: 50 μM PSP2, 50 mM NADH, 100 mM Tris-HCl pH 7.0 buffer. For each photoperiod, the sample was first irradiated with a 405nm laser (100mW / cm ² ) for 10 minutes, and then the absorbance at 525nm (which represents the formation of PSP2 ·, corresponding to one of the three isoabsorbance points), and then Incubate for 20 minutes in the dark before the start of the next light cycle. The reversible formation of PSP2 · indicates that although PSP2 · can react with oxygen (this is a common characteristic of almost all super-reducing free radicals), the reaction returns to the ground state PSP2 without destroying the chromophore. Since various carbon dioxide reducing agents are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of the catalyst.

Figure 10. LC-MS spectrum of a PSP2 monocysteine mutant modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1).

a, MS characterization of PSP1-26C. Calculated molecular weight: 27604 Da; measured molecular weight: 27606 Da.

b, MS characterization of PSP1-26C modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide. Calculated molecular weight: 27893 Da; found molecular weight: 27892 Da.

c, MS characterization of PSP2-95C modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide. Calculated molecular weight: 27899Da; measured molecular weight: 27899Da.

d. PSP2-95C 93Y97Y modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide. Calculated molecular weight: 28030 Da, measured molecular weight: 28025 Da.

e. MS characterization of PSP2 modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide. Calculated molecular weight: 27646 Da; measured molecular weight: 27743 Da. The measured molecular weight is basically consistent with the calculated molecular weight, which indicates that when a single cysteine mutation is not introduced into PSP2, N- (2,6,2-terpyridin-4-yl) -iodoacetamide cannot be conjugated to PSP2. Further, this can also prove that for PSP2 variants that introduce a single cysteine residue, N- (2,6,2-terpyridin-4-yl) -iodoacetamide is specifically conjugated to the cysteine Amino acid residues.

Figure 11. UV spectra of terpy, Ni (II) terpy, PSP2-95C-terpy, PSP2-95C-Ni (II) terpy, and purified PSP2-95C-Ni (II) terpy. These results show that when 8 μM PSP2-95C-terpy is mixed with 2 equivalents of Ni (II), the PSP2-95C-Ni (II) terpy complex is quantitatively formed as shown by the 335 nm peak.

Figure 12. The distances between the β-carbon atoms of residues 147, 151, 95, 155, 26 and the PSP2 chromophore are 6.0, 10.2, 11.9, 17.6,

Figure 13. Before (a) and after (b), using 130mW · cm ^-2 Xe light decomposition equipped with AM 1.5 filter, containing 0.4 μM PSP2T1, 0.8 μM NiClO ₄ , 1 mM BIH (ie, 4- (2, TEM image of a reaction solution of 3-dihydro-1H-benzo [d] imidazol-2-yl) benzene-1,2-diol), 1 mM NaHCO ₃ .

Figure 14. Amino acid sequences of sfYFP (SEQ ID NO: 1) and various sfYFP variants constructed according to the present invention (SEQ ID NOs: 2, 4, 6, 8, 10), of which the "*" at position 66 of the variant Represents BpA.

Sequence listing description

SEQ ID NO: The following is a description of

SEQ ID NO: 1 amino acid sequence of superfolded yellow fluorescent protein (sfYFP)

SEQ ID NO: 2 amino acid sequence of sfYFP-BpA66

SEQ ID NO: 3 Nucleotide sequence of sfYFP-BpA66 expressed in E. coli

SEQ ID NO: 4 amino acid sequence of sfYFP-BpA66-Phe203 (PSP1)

SEQ ID NO: 5 Nucleotide sequence of sfYFP-BpA66-Phe203 (PSP1) expressed in E. coli

SEQ ID NO: 6 amino acid sequence of sfYFP-BpA66-Asp203 Glu148 (PSP2)

SEQ ID NO: 7 nucleotide sequence of sfYFP-BpA66-Asp203 Glu148 (PSP2) expressed in E. coli

SEQ ID NO: 8 amino acid sequence of PSP2-95C

SEQ ID NO: 9 Nucleotide sequence of PSP2-95C expressed in E. coli

SEQ ID NO: 10 amino acid sequence of PSP2-95C93Y97Y

SEQ ID NO: 11 Nucleotide sequence of PSP2-95C93Y97Y expressed in E. coli

SEQ ID NO: 12 Nucleotide sequence of superfolded yellow fluorescent protein (sfYFP) expressed in E. coli

It should be noted that because benzophenone-alanine (BpA) is not a natural amino acid, such artificial amino acids cannot be displayed when preparing a sequence list in a computer-readable form. Therefore, SEQ ID NOs in the sequence list: 2, 4 In the sfYFP variant sequences shown in Figures 6, 6, 8, and 10, Tyr is still displayed at position 66, and the inventor noted in <223> that "mutation of Tyr at position 66 to benzophenone-alanine (BpA) "Information. Those skilled in the art can understand the sfYFP variant sequences shown in SEQ ID NOs: 2, 4, 6, 8, and 10 according to the information described in this specification in combination with the variant sequences listed in FIG. 14, which is actually 66th Benzophenone-alanine (BpA).

detailed description

The present invention is further described below with reference to specific embodiments, but those skilled in the art should understand that the present invention is not limited to these specific embodiments.

Unless otherwise stated, the reagents, plasmids, etc. used in the examples can be purchased from commercially available channels.

Materials and Method

material

2-Amino-3- (4-benzoylphenyl) propionic acid (BpA, benzophenone-alanine) was purchased from Amatek Scientific company (Suzhou, China). 4-Amino-2,6,2-terpyridine was purchased from Shanghai UCHEM Company (Shanghai, China). BIH (ie, 4- (2,3-dihydro-1H-benzo [d] imidazol-2-yl) benzene-1,2-diol) was synthesized according to the method in the reference ⁴¹ . All other chemicals were purchased from Sigma-Aldrich or J & K chemical and used without further purification. Silica gel chromatography was performed using silica gel 60 (230-400 mesh, purchased from Inokay Corporation). PCR reagents, T4 DNA ligase and restriction endonuclease were purchased from Fermentas. Ni-NTA affinity purification reagents and purification columns were purchased from Qiagen. The pEVOL-BpARS plasmid was purchased from Addgene (Plasmid # 31190). The primers and mutated genes used were synthesized by Sangon Biotech.

Analytical method

¹ H and ¹³ C NMR spectra were recorded on a Bruker AMX-500 instrument, and the chemical migration of tetramethylsilane was used as a reference migration. All ¹ H NMR spectra are reported in parts per million (ppm) and are measured relative to the DMSO signal (2.5 ppm). ^{The 13} C NMR spectrum is reported in ppm relative to the residual DMSO (40 ppm). The mass spectra of the chemicals were run on a Waters LC-MS (Waters ACQUITY QDa) equipped with a single quadrupole mass detector and an electrospray ion source. The mass spectrum of the protein was run on an Agilent 6100 series single quadrupole mass spectrometer (Agilent Technologies). Protein purification was performed on the AKTA UPC 900FPLC system (GEhealthcare). The absorption spectrum was recorded at room temperature using an ultraviolet-visible mass spectrometer (Agilent 8453, Agilent technologies, CA, USA). Fluorescence spectra were recorded on a microplate reader equipped with Varioskan Flash SkanIt software 2.4.3RE (Varioskan Flash, Thermo Fisher Scientific Inc). The fluorescence attenuation measurement was performed with a time-correlated single photon counting (TCSPC) fluorescence spectrometer (FL900 Edinburgh instruments Ltd.). Instantaneous absorption spectra with nanosecond time resolution were detected using an Edinburgh LP980 spectrometer (Edinburgh Instruments Ltd.) set up with nanosecond flash photolysis. Cyclic voltammetry (CV) measurements were performed using a CH instrument 600D electrochemical system (CH Instrument, China). Gas chromatography (GC) was performed with an SRI Multi-Gas Analyzer (SRI Instruments, Model 8610C) equipped with a TCD and HID detector. Circular dichroism (CD) spectra were recorded with a circular dichroism (Applied Photophysics Ltd, Chirascan Plus). The above experiments were performed at the Institute of Biophysics, Chinese Academy of Sciences. ESR spectra were recorded with a 154 Bruker EMX-plus X-band spectrometer (Institute of Chemistry, Chinese Academy of Sciences, Beijing). Transmission electron microscope (TEM) pictures were taken with a JEM-2100F electron microscope (Analytical Center, Department of Chemistry, Tsinghua University).

Example 1. Synthesis and characterization of corresponding compounds

The following compounds are synthesized according to the following reaction route, and are used to detect the reduction potential of free radicals formed by the genetically encoded artificial photosynthesis protein prepared in the present invention during photoreaction.

Unless otherwise specified, the reagents used in the following synthesis reactions were purchased from B & W Chemical Reagent Company.

Synthesis of (E) -4- (4-benzoylbenzylidene) -1,2-dimethyl-1H-imidazole-5 (4H) -one (BpAChm) (6)

(1) Synthesis of 4-((2-methyl-5-oxooxazole-4 (5H) -ylidene) methyl) benzaldehyde (3)

A mixture of 1 (23.6 g) and NaOAc (16.4 g) in Ac ₂ O (100 mL) was stirred at room temperature for 1 hour, and then 2 (26.8 g) was added. The mixture was stirred at room temperature for 2 hours and then at 65 ° C overnight. After cooling to room temperature, H ₂ O (1 L) was added to the mixture. The mixture was stirred at room temperature for 1 hour and then filtered. The obtained solid was washed with H ₂ O (1 L) and MeOH (100 mL) and then dried in vacuo to give 3 (32 g) as an orange solid, which was used directly in the next step without further purification.

(2) Synthesis of 4-((1,2-dimethyl-5-oxo-1H-imidazole-4 (5H) -ylidene) methyl) benzaldehyde (4)

To a solution of 3 (32 g) in EtOH (100 mL) was added NH ₂ Me (40% in EtOH, 100 mL). The mixture was stirred at room temperature for 1 hour and then at 65 ° C overnight. After removing the solvent, the residue was purified by silica gel chromatography, eluting with PE to PE: EA = 1: 1 (v / v), to give 4 (3.6 g) as a pale yellow solid.

Wherein PE is petroleum ether and EA is ethyl acetate.

(3) Synthesis of 4- (4- (hydroxy (phenyl) methyl) benzylidene) -1,2-dimethyl-1H-imidazole-5 (4H) -one (5)

To a solution of 4 (3.6 g) in THF (100 mL), PhMgBr (in Et ₂ O, 3M, 5 mL) was added at -78 ° C. The mixture was stirred at -78 ° C for 1 hour, and then further stirred at 15 ° C for 1 hour. To the mixture was added MeOH (50 mL), and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, eluting with PE to PE: EA = 1: 1 (v / v), to give 5 (2.1 g) as a pale yellow solid.

Wherein PE is petroleum ether and EA is ethyl acetate.

(4) Synthesis of 4- (4-benzoylbenzylidene) -1,2-dimethyl-1H-imidazole-5 (4H) -one (6)

To a solution of 5 (2.1 g) in DCM (100 mL) was added Dess-Martin (5 g, purchased from Alfa Chemical Reagent Company). The mixture was stirred at rt for 1 h, then washed with saturated NaHCO ₃ (300mL) quench the reaction. The organic layer was separated and washed with saturated Na ₂ SO ₃ (100 mL), and then concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with PE to PE: EA = 1: 1 (v / v), to give crude product 6 (1.1 g). It was further purified and titrated with EA: MeOH = 9 mL: 1 to give pure product 6 (0.75 g) as a pale yellow solid.

MS (ESI): C ₁₉ H ₁₆ N ₂ O ₂ requires m / z to calculate mass: 304.12, found [M + 1] ⁺ m / z 305.02; [M + Na] ⁺ m / z 327.02. ¹ H-NMR ( 500MHz, DMSO-d6) 8.34 (s, 1H), 8.32 (s, 1H), 7.77 (m, 4H), 7.75 (m, 1H), 7.56 (m, 2H), 7.02 (s, 1H), 3.1 ( s, 3H, -CH ₃ ), 2.37 (s, 3H, -CH ₃ ); ¹³ C-NMR (500 MHz, DMSO-d6) δ 195.7, 170.3, 166.7, 141.0, 138, 4, 137.6, 137.2, 133.3 , 132.1, 130.2, 130.1, 129.1, 123.3, 26.8, 15.9.

Synthesis of N- (2,6,2-terpyridin-4-yl) -iodoacetamidoterpyridine (7)

(1) Synthesis of N- (2,6,2-terpyridin-4-yl) -chloroacetamide

4-amino-2,6,2-terpyridine (240 mg, 1 mmol, purchased from Shanghai UCHEM Company) was dissolved with a mixture solvent (THF / MeCN = 1 mL: 1 mL) consisting of tetrahydrofuran and acetonitrile. To the solution was added triethylamine (500 μL, 5 eq.), And stirred under a nitrogen atmosphere for 1 hour. then. Chloroacetyl chloride (200 μL in 1 mL MeCN) was added dropwise. The mixture was stirred for 2 hours. The resulting solution was extracted with ethyl acetate (EtOAc), washed with 5% NaHCO ₃ solution. The organic phase was collected, dried over anhydrous Na ₂ SO _4, and evaporated to give a brown solid (300mg). This product was used in the next step without further purification ([M + 1] ⁺ m / z 325).

(2) Synthesis of N- (2,6,2-terpyridin-4-yl) -iodoacetamide

N- (2,6,2-terpyridin-4-yl) -chloroacetamide (300 mg) was dissolved in a mixed solvent consisting of tetrahydrofuran and acetonitrile (THF: MeCN = 1 mL: 10 mL). Potassium iodide (500 mg) was added, and the mixture was refluxed at 90 ° C for 1 hour. The obtained suspension was concentrated and separated through a silica gel column (eluent CH ₂ Cl ₂ : CH ₃ OH = 20 mL: ₁ mL) to obtain the product (150 mg) as a yellow powder in a yield of 35%.

¹ H-NMR (500 MHz, DMSO-d6) 8.70 (d, 2H), 8.66 (s, 2H), 8.58 (d, 2H), 8.00 (t, 2H), 7.49 (t, 2H), 3.89 (s, 2H); ¹³ C-NMR (500MHz, DMSO-d6) δ 168.52, 156.23, 155.10, 149.56, 148.18, 138.07, 125.03, 121.33, 110.51, 1.43; MS (ESI): calculated for C ₁₇ H ₁₃ IN ₄ O Quality needs m / z: 416.01, measured [M + 1] ⁺ m / z 417.02.

Synthesis of N- (2,6,2-terpyridin-4-yl) -iodoacetamide nickel (II) complex

A solution of Ni (ClO ₄ ) ₂ .6H ₂ O in acetonitrile (500 μL, 20 mM) was added to a tube containing N- (2,6,2-terpyridin-4-yl) -iodoacetamide (10 μmol). The mixture was diluted to 1 ml with water (500 μL) and sonicated for 10 minutes to obtain a clear yellow-orange stock solution.

The calculated mass of C ₃₄ H ₂₆ NiI ₂ N ₈ O ₂ requires m / z: 890.96, measured [M] ²⁺ m / z 445.46.

Example 2.Construction of sfYFP mutant

All vectors expressing fluorescent protein (FP) variants described in the present invention were cloned and expressed using the pET22b (+) vector (purchased from General Biological Systems (Anhui) Co., Ltd.). The PCR reaction (50 μL) contained 10 pM primers, 50 ng of template DNA, 1 × high-fidelity DNA polymerase buffer, 1 unit of high-fidelity polymerase (Fermentas), 0.2 mM dNTP, and 1.5 mM MgCl ₂ . DNA amplification was performed using a DNA thermal cycler: initial denaturation (94 ° C, 1min); followed by 30 strand reaction cycles: 94 ° C 1min, 60 ° C 1min, 68 ° C 1min; and finally extension at 68 ° C for 10min.

A vector pET22b (+) containing a superfolder yellow fluorescent protein (sfYFP, amino acid sequence shown in SEQ ID NO: 1) coding sequence (SEQ ID NO: 12) was used as a template for generating different sfYFP mutants. All constructs and their mutagenesis were verified by DNA sequencing analysis.

The construct pET22b-sfYFP (where the sfYFP coding sequence was synthesized by Universal Biosystems (Anhui) Co., Ltd.) and the BpA tRNA synthetase plasmid pEVOL-BpARS (the plasmid was purchased from Addgene (Plasmid # 31190), please refer to reference 38 for the method of use -39) Co-transformed into E. coli BL21 (DE3) strain for unnatural amino acid incorporation. Specifically, the construct pET22b / sfYFP-TAG66 mutant and pEVOL-BpARS were co-transformed into the E. coli BL21 (DE3) strain. The pEVOL-BpARS plasmid carries a BpA-selective Methanococcus jannaschii tyrosyl tRNA synthetase and a MjtRNA tyrosyl amber suppressor tRNA (MjtRNA _Tyr ^CUA ), thereby allowing the first to the sfYFP mutant The 66-site specific incorporation of BpA, the resulting mutant protein was named sfYFP-BpA66, and its amino acid sequence is shown in SEQ ID NO: 2.

On this basis, by changing the corresponding codon nucleotides of the sfYFP coding sequence in the expression vector, the 203th tyrosine (Tyr) in sfYFP was further mutated to phenylalanine (Phe), the double mutant sfYFP -BpA66-Phe203 is named PSP1, and its amino acid sequence is shown in SEQ ID NO: 4. Furthermore, a triple mutant sfYFP in which tyrosine (Tyr) at 203 of sfYFP was mutated to aspartic acid (Asp) and histidine (His) at 148 was mutated to glutamic acid (Glu). -BpA66-Asp203 Glu148 is called PSP2, and its amino acid sequence is shown in SEQ ID NO: 6.

On the basis of PSP2, the 95th glutamic acid (Glu) was mutated to Cys (C) to obtain a PSP2-95C mutant, whose amino acid sequence is shown in SEQ ID NO: 8. This mutant was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- (2,6,2-terpyridine-4) -Yl) -iodoacetamide is specifically conjugated to cysteine at position 95). The final conjugate obtained in the presence of divalent nickel ions is named PSP2T1, and it was found that PSP2T1 has a higher carbon dioxide Reducing activity.

In order to study whether the presence of a local proton donor can improve the catalytic efficiency, the inventors mutated both the 93th valine (Val) and 97th threonine (Thr) to tyrosine based on the PSP2-95C mutant Acid (Tyr, Y) to obtain mutant PSP2-95C93Y97Y, the amino acid sequence of which is shown in SEQ ID NO: 10. This mutant was modified with N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7 synthesized in Example 1) (N- (2,6,2-terpyridine-4) -Yl) -iodoacetamide is specifically conjugated to cysteine at position 95), and the final conjugate obtained in the presence of a divalent nickel ion is named PSP2T2, which shows a significantly increased number of carbon monoxide conversions (TON) (Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3).

For protein expression, a single colony that had been transformed with the corresponding recombinant expression vector was placed in LB supplemented with ampicillin (100 μg / mL, purchased from Sigma-Aldrich) and chloramphenicol (25 μg / mL, purchased from Sigma-Aldrich). The medium (4 mL, purchased from Sigma-Aldrich) was grown overnight at 37 ° C. 1 mL of the overnight culture was inoculated into 100 mL of liquid LB medium supplemented with ampicillin (100 μg / mL) and chloramphenicol (25 μg / mL) and BpA (1 mM). The cells were then cultured at 37 ° C to an OD 600 of 1.1, followed by addition of 0.02% arabinose (purchased from Sigma-Aldrich) and 1 mM isopropyl β-D-1-galactopyranoside (IPTG, purchased from Sigma- Aldrich) induces protein expression. Continue incubation for 4-12 hours, collect cells and freeze at -70 ° C, ready for protein purification.

For protein purification, cells were suspended in lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 10 mM imidazole) and lysed by sonication. After centrifugation, the supernatant was loaded onto a Ni-NTA column (Histrap 5ml, GE Healthcare). Wash the column twice with 5 ml of washing buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 50 mM imidazole), and then elute with an elution buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 250 mM imidazole). Captured protein.

During the crystallization, a solution of each purified protein in a buffer containing 20 mM HEPES-NaOH, pH 7.5, and 10 mM β-ME was concentrated to 10 mg / mL. 0.5 mg / mL trypsin (TPCK-treated) was added and incubated at 37 ° C for 1.5 hours. The degradation reaction was blocked by adding 1 mM PMSF (phenylmethylsulfonyl fluoride, purchased from Sigma-Aldrich). The above mixture was reloaded onto a Ni-NTA agarose affinity resin (purchased from Sigma-Aldrich) to remove undigested proteins containing the complete HIS6 tag. The digested protein was purified by Sephadex gel column chromatography (Superdex 75 10 / 300GL; GE Healthcare) into a buffer containing 20 mM HEPES-NaOH, pH 7.5, and concentrated to ~ 30 mg / mL, and examined by SDS-PAGE. By mixing 1 μL protein sample (20mg / mL) with an equal volume of crystallization buffer (ground state: 25% PEG3350, 0.2M MgCl ₂ , 0.1M Bis-Tris pH 5.5; free radical state: 15% PEG3350, 0.1M malic acid, pH 6.5) Mixing, using a sitting-drop vapor diffusion method at 16 ° C for about one week. The crystals were then quickly frozen in liquid nitrogen.

Regarding the determination of the crystal structure of the mutant, the diffraction data was collected by Shanghai Synchrotron Radiation Facility (SSRF) using beamlines B17U or BL18U. Data processing and reduction were performed using HKL2000 package. Use the sfYFP (PDB code: 1F0B) atomic coordinates as the retrieval model, and use the CCP4 suite (a macromolecular structure crystal structure analysis software, Collaborative Computational Proiect No.4 Software) for Macromolecular X-Ray Crystallography, http: //www.ccp4 .ac.uk /) Molrep performs molecular replacement to analyze the structure of PSP2 · (radical state).

Data collection and structural refinement statistics are summarized in Table 1 below. A schematic diagram of the protein structure was generated using PyMOL (http://www.pymol.org). The atomic coordinates and structural factors of the PSP2 · (radical state) crystal structure have been registered in the Protein Data Bank (PDB codes: 5YR3).

Table 1. Statistical data of X-ray diffraction of PSP2 ·.

An Agilent 8453 UV-visible spectrophotometer was used to detect the UV-Vis spectra of various PSP mutant proteins at room temperature and determine the pKa of PSP2 ·. UV-Vis spectroscopy and pKa data can prove that the PSP mutant protein is converted to a deprotonated state under alkaline conditions.

Using an Agilent 8453 UV-visible spectrophotometer, the formation of PSP2 radicals (PSP2 ·) was reversibly detected at room temperature. In the presence of 50 mM NADH, 50 μM PSP2 protein in 100 mM Tris-HCl pH 7.0 buffer was irradiated with a 405 nm laser for 10 minutes, and then using an Agilent 8453 UV-visible spectrophotometer (quartz cup, 100 μL, 1 cm path) UV-Vis spectra were recorded at room temperature. For each photoperiod, the sample was first irradiated with a 405nm laser (100mW / cm ² ) for 10 minutes, and then the absorbance at 525nm (which represents the formation of PSP2 ·, corresponding to one of the three isoabsorbance points), and then Incubate for 20 minutes in the dark before the start of the next light cycle. The reversible formation of PSP2 · indicates that although PSP2 · can react with oxygen (a common characteristic of almost all super-reducing free radicals), the reaction returns to PSP2 without destroying the chromophore. Since various carbon dioxide reducing agents are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of the catalyst.

The reduction potential was measured by cyclic voltammetry (CV). The reduction potential data can indirectly prove the reducing power of PSP2 radicals. Measurements were performed with CH Instruments 600D potentiostat (instrument purchased from Shanghai Chenhua Instrument Co., Ltd.). A 2 mM BpAChm solution in a 0.1 M NBu ₄ PF ₆ DMF solution was placed in a 3-electrode chamber with an Au working electrode, an Ag / AgCl reference electrode, and a Pt auxiliary electrode at 4 ° C. Prior to measurement, the system was purged with Ar for 15 minutes. The CV parameters are as follows: scan rate: 10mV / s; sample interval: 1mV; sensitivity: 10μA / V; quiet time: 4s; temperature: 0 ° C.

Electron spin resonance (ESR) experiments were performed to further characterize the production of free radical PSP2 ·. In the presence of 50 mM NADH, 45 μM PSP2 in 100 mM Tris-HCl pH 8.0 buffer was propagated with 405 nm laser radiation 10, and the yield of PSP2 free radicals was quantitatively determined by UV-Vis spectroscopy as 12%. Before the 405nm laser irradiation, the PSP2 protein solution appeared light yellow, and after the 405nm laser irradiation, the protein solution became dark red, indicating that free radicals were formed. X-band ESR spectra were then recorded on a Bruker E500 spectrometer at room temperature for dark red light-treated protein samples. ESR capture parameters are as follows: adjustment frequency: 30-100kHz; microwave power: 0.05-10mW; adjustment amplitude: 2G.

In order to determine the absorption spectrum and lifetime of various intermediates after PSP photoexcitation, a nanosecond flash photolysis device using an Edinburgh LP980 spectrometer (Edinburgh Instruments Ltd.) combined with a compact Q-switched Nd: YAG laser (Q-smart 850, Quantel, France) measures instantaneous absorption spectra with nanosecond time resolution. The probe is a 150W pulsed xenon arc lamp for kinetic and spectral measurements from a few nanoseconds to 1ms. Photolysis of the samples was achieved using a single-flash laser excitation at 355 nm (1 Hz, 10 mJ / pulse, 50 mm ² spot area, fwhm ≈ 7 ns). The probe light comes from a 450W pulsed xenon lamp. A single detector (PMT R928P) was used to record transient signals, an oscilloscope was used to record kinetic traces, and an ICCD detector was used to record time-resolved spectra. Data were analyzed using LP900 software. A sample having an absorbance of 0.3 OD at a wavelength of 355 nm was first degassed with Ar for about 10 minutes before measurement.

The attenuation curve is fitted to the following equation, and the obtained lifetime values are listed.

The constant rate is calculated as follows:

Example 3. Activity detection of sfYFP mutant

3.1 Terpyridine modification of PSP2 monocysteine mutant

A monocysteine mutant of PSP2 (refer to Table 3, 50 μM) in a reaction buffer (150 mM Tris-HCl buffer pH 8.8, 30% DMF) at room temperature with 100 μM tris (2-carboxyethyl) Phosphine (TCEP) treatment for 5 minutes. Then, a labeling reaction was performed at room temperature for 12 hours by adding 250 μM N- (2,6,2-terpyridin-4-yl) -iodoacetamide (the compound 7 synthesized in the example). After terpyridine modification, unbound terpyridine molecules were removed by dialysis against 10 mM Tris-HCl pH 8 buffer (Amicon Ultra-0.5 Centrifugal Filter Unit 3K, at least 3 times). The obtained mutants were then analyzed by LC-MS. Figure 11 shows the quantitative formation of the PSP2-95C-terpy complex.

3.2 Photocatalytic carbon dioxide reduction

Photocatalytic carbon dioxide reduction was performed in a glass headspace bottle (total volume 10 ml) sealed with a corrugated stopper. For a typical reaction, the reaction solution volume is 200 μL: 100 mM Tris-HCl buffer (pH 8.0, 50% DMF, to increase 4- (2,3-dihydro-1H-benzo [d ] Imidazol-2-yl) benzene-1,2-diol (BIH) derivative solubility) terpyridine nickel complex modified 40 μM PSP2 monocysteine mutant protein (for example, PSP2T1 or PSP2T2) and 80 μM Ni (ClO ₄ ) ₂ , 100 mM NaHCO ₃ , 100 mM 4- (2,3-dihydro-1H-benzo [d] imidazol-2-yl) benzene-1,2-diol (BIH) sacrificial reducing agent (SR) were added to the glass headspace vial together. The sample was bubbled with argon (Ar) for 10 minutes, and then irradiated with a 300W Xe lamp (MICROSOLAR300, Beijing Perfilai Technology Co., Ltd.) with an AM 1.5 filter to simulate the solar spectrum. A cutoff filter (UVCUT400) is used to achieve visible light (λ> 400nm) irradiation. Gas chromatography (GC) (SRI instruments, 8160C GC) was used to analyze the photocatalytic gas generation rate.

3.3 Calculating Photons (Photon Hits / Molecules / Sec) ⁴²

Photon hits / molecules / second were calculated according to the method of reference 42 (see pages 60-61 for details).

The photon flux or intensity I is calculated by:

I = E * N _A

Where I is the number of photons per unit surface area per second, E is the number of Einsteins per unit area per second, and N _A is the Avogadro constant with a value of 6.02 * 10 ²³ .

By replacing the absorbed sunlight intensity (400-450nm) with PSP2, the inventors obtained: I = 1.32 × 10 ²⁰ photons m ^-2 s ^-1 ; this corresponds to 1.32 photons

For the sfYFP protein, the structure is roughly square, and each side of the square is long

It is illuminated by about 16 photons per second, but not all of these photons are absorbed. To determine how many photons are absorbed, the target size of the photosensitizer should be calculated according to the following equation:

σ = 2303ε / N _A

Where σ is the target size, ε is the molar extinction coefficient, and N _A is the Avogadro constant.

The extinction coefficient at the maximum absorption wavelength of 375 nm is 23 mM ^-1 cm ^-1 . The average extinction coefficient in the absorption region (400 nm to 450 nm) is about 3.65 mM ^-1 cm ^-1 . The average size σ of the BpA-containing protein is 1.39 × 10 ^-17 cm ² , that is,

Then the photon hit / molecule / second is I × σ photons s ^-1 , Hits = I × σ = 1.32 photons

3.4 Determine the quantum efficiency value

The following equation is used to calculate the quantum efficiency (QE) value of the catalytic photooxidation reaction:

TON _co represents the carbon monoxide conversion number, and Hits is the Hits calculated in Section 3.3.

Using the ideal gas law regarding the system volume of the inventors, the carbon monoxide concentration (in ppm) detected by gas chromatography was converted to the total number of moles of carbon monoxide produced. Because it takes 2 electrons to generate one molecule of carbon monoxide, a factor of 2 is included in the calculation. To determine the flux of incident photons, the photon wavelength (400-450 nm) was determined from the absorbance of the protein mutant containing BpA, and the flux was calculated.

The calculation is based on the number of photons absorbed by PSP2T2 after 3 hours photolysis, and the calculated quantum efficiency (QE) is 2.6%.

With regard to the total number of incident solar photons, the quantum efficiency (QE) based on incident photons is calculated according to the method described in reference 43:

Here, the incident photons number can be calculated from the incident photon flux 1.2 × 10 ²¹ photons · cm ^-2 · h ^-1 (at 130mW cm ^-2 ), and in the case of the present inventors' study, the irradiation area It is 1 cm ² . After 3 hours photolysis, 0.2 μmol of carbon monoxide (CO) was produced, and the total volume of the reaction system was 0.2 ml. Based on the total number of irradiated photons, the QE calculated for the PSP2T2 system was 0.0067%.

Results and discussion

In order to design a photocatalytic carbon dioxide reductase, the present inventors first converted a fluorescent protein (FP) ^13-14 into a photosensitizer protein (PSP) (Figure 1). In order to achieve this goal, the following conditions must be met: (1) the PSP must be able to effectively absorb visible light; (2) when absorbing photons, the PSP must be converted into a photoexcited state (PSP *) that can exist for a longer time, thereby promoting Electron transfer reaction, which leads to the formation of PSP radicals (PSP ·); (3) PSP · must be a strong reducing agent that can drive the reduction of carbon dioxide reduction catalysts. Because carbon dioxide is highly inert, the reduction of carbon dioxide reduction catalysts usually requires a high overpotential ^5. Since natural fluorescent proteins usually only have a nanosecond excited state lifetime of ¹⁴ and exist for too short a time to allow long-range electron transfer, the present inventors expressed ^15-16 benzophenone-alanine (BpA) by using the genetic code. (Figure 1b) replaces the chromophore residue Tyr66 in the superfolder yellow fluorescent protein (sfYFP, Figure 2-3) to modify the FP chromophore. sfYFP produces a highly fluorescent p-hydroxybenzylidene-5-imidazolinone (p-HBI) species through the spontaneous catalytic conversion of its tripeptide Gly65-Tyr66-Gly67. Benzophenone is known to nearly 100% quantum efficiency from the excited singlet state to the triplet state crossing lines, having a lifetime of the triplet state is ⁵ times the original 10, which allows the occurrence of sacrificial reducing agent (SR) reduction ^{17- 21} . The present inventors envisage that in the sfYFP mutant containing BpA66, the tripeptide Gly65-BpA66-Gly67 can be spontaneously and catalytically converted to (E) -4- (4-benzoylbenzylidene) -1,2-di The chromophore of methyl-1H-imidazol-5 (4H) -one (BpAChm, structure shown in Figure 1f) should cross to the triplet state with high quantum efficiency.

The inventors envisage that once the FP chromophore is photochemically reduced, a color change should be observed. To test this assumption, the inventors first incorporated BpA into position 66 of sfYFP, and irradiated the mutant protein with a 405 nm laser (100 mW / cm ² ) in the presence of 10 mM sodium dithionite. Since the 203 amino acid residue of sfYFP is tyrosine, it undergoes a rapid electron transfer process with the PSP chromophore due to the formation of a pi-stack. Therefore, PSP cannot be detected after light irradiation. . This may be due to the electron transfer from Tyr203 to PSP * and the consequent rapid charge binding. Then, the inventors mutated Tyr203 to Phe. Unexpectedly, the sfYFP-BpA66-Phe203 double mutant (PSP1, SEQ ID NO: 4) changed color from yellow to red 30 seconds after laser irradiation (Figure 1b, Figure 5). Irradiation leads to the disappearance of 390nm peak at 555nm and 765nm with two new peaks appear, indicating that photochemical reduction reaction has taken place ^22. The resulting photochemical product is called PSP1 ·.

Since dithionite is a strong reducing agent that is not produced in living cells (at pH 7, E ^o = -436mV), the inventors then investigated whether a weaker biologically related reducing agent can promote the photochemical reduction of PSP1 . The inventors found that sodium ascorbate cannot drive the photochemical reduction of PSP1. In order to increase the oxidation potential of PSP * so that it can receive electrons from bio-related sacrificial reducing agents (SR) to generate PSP ·, the inventors mutated residues Phe203 and His148, and the residues near the PSP1 chromophore were mutated to Aspartic acid, glutamic acid or lysine. Mutation of these residues into charged residues can significantly adjust the PSP chromophore reduction potential through electrostatic and hydrogen bonding interactions ^1,2 . These mutant PSP proteins were then irradiated with a 405 nm laser in the presence of ascorbate, and sfYFP-BpA66-Asp203Glu148 (PSP2, SEQ ID NO: 6) was found to change from yellow to red after 405 nm laser irradiation (Figure 6). These results indicate that PSP2 utilizes a bio-related sacrificial reducing agent to perform an effective photochemical reduction reaction to produce PSP2 ·.

Then, the inventors performed UV-Vis titration of PSP2 · under different pH conditions. As shown in Figs. 1c and 1d, although PSP2 · has a strong peak at 500nm at a near neutral pH, new peaks appear at 555nm and 765nm after illumination, and the new peak increases with increasing pH. These isoabsorption points were observed at 525 nm, 680 nm, and 730 nm, respectively, which indicates that only two chemical species are present during the titration. These results indicate that PSP2 · exists as a neutral radical (pKa = 10.6) at a neutral pH. In contrast, the UV-Vis spectrum of PSP1 · remained unchanged from pH 6 to 10, which indicates that PSP1 · is an anionic group at neutral pH (Figures 6 and 7). At pH 6-11.8, the CD spectrum of PSP2 showed no change in the secondary β-sheet structure, indicating that the protein remained correctly folded throughout the pH titration process (Figure 8). These results indicate that PSP2 * is an oxidant superior to PSP1 *. Residues Asp203 / Glu148 can transfer protons to PSP2 * at a rate similar to electron transfer. This proton coupled electron transfer (PCET) ^23-24 (proton coupled electron transfer (PCET)) avoids the generation of high-energy anionic group intermediates, reduces the PSP · state energy, and therefore increases the energy difference between PSP * and PSP ·, which The electron transfer rate from SR to PSP * will be accelerated to form PSP ·.

To further characterize the generation of PSP2 radicals by photoexcitation, the inventors collected X-band electron spin resonance (ESR) data, which showed that after 405nm laser irradiation (not before irradiation) The strong peak at g = 2.006 and the peak-to-peak width of ~ 22Gauss (Figure 1e), which proves that a typical organic radical group is formed in PSP2 ·.

In order to characterize the reduction potential of PSP2, the inventors performed electrochemical detection of proteins in aqueous solution. However, due to the low reduction potential of PSP2, no significant signal corresponding to protein reduction was observed in the reduction potential range before water redox. Then, the inventors synthesized a small molecule BpAChm that mimics the PSP2 chromophore (Figure 1f). Cyclic voltammetry in DMF solvent (CV) experiments show, BpAChm having two reduction peaks at -1.46V and -2.05V, which 1e and 2e BpAChm with corresponding reduction ²² (FIG. 1f). Since DMF is not a good mimic of the internal environment of proteins, the value of E ~ -1.46V cannot accurately represent the same reduction potential of PSP2. In this case, the inventors used the strong reducing agent rhenium (II) diethylenetriamine pentaacetate (Eu (II) -DTPA, purchased from Sigma-Aldrich) (at pH 8, E ⁰ ′ = -1.14 V) ²⁵ , check whether PSP2 can be restored. The UV-Vis spectrum showed that even when an 80-fold excess of Eu (II) -DTPA was used as the reducing agent, no PSP2 · absorption peak appeared (Fig. 6d). Based on these data, the inventors can speculate that the reduction potential of PSP2 · is less than -1.14V.

In order to test whether PSP2 · is sensitive to oxygen, the present inventors first produced PSP2 · by light irradiation, and then incubated it in the presence of air in 100 mM Tris-HCl pH 7.0 buffer for 20 minutes. As shown in Fig. 9, PSP2 · reacts with oxygen to generate PSP2. This light cycle can be repeated multiple times. The reversible formation of PSP2 · indicates that the reaction of PSP2 · with oxygen will not irreversibly destroy the chromophore. Since many carbon dioxide reduction catalysts are known to be irreversibly destroyed by oxygen, this characteristic is important for the robustness of PSP-based catalysts.

In order to characterize the structural changes due to photochemical reduction, the inventors used X-ray crystallinity to

The resolution determines the structure of PSP2 ·. Under the condition of 405nm laser irradiation, the acquisition of X-ray diffraction data was completed within 3 minutes. The inventors have confirmed that the crystals remain dark red throughout the data acquisition process (Figure 2a / b). Prior to photochemical reduction, the two benzene rings in residue BpA66 adopt a twisted conformation with a dihedral angle of 58 ° (Figure 2c). Similarly, the dihedral angle between two benzene rings in benzophenone is 56 ° ²⁶ , which indicates that the two benzene rings do not form a conjugated π-electron system. However, in the free radical state (PSP2 ·), one benzene ring in BpA66 proceeds

The apparent dihedral rotation (Figure 2e) reduces the dihedral angle between the two benzene rings by 29.1 ° (Figure 2d), resulting in the formation of an extended conjugated π-electron system and a significantly red-shifted UV-Vis spectrum. To the knowledge of the inventors, this is the first report on the crystal structure of protonated and neutral benzophenone radicals. Detailed structural information about the super-reducing benzophenone radicals caged in the PSP2 · protein rigid structure cage provides the necessary understanding for using this powerful reagent to drive challenging enzyme reactions.

In order to convert the PSP into a photo-sensitive carbon dioxide reductase, the inventors used a nickel-terpyridine complex ²⁷ (E ⁰ = -1.0 V (Ni (II / I)); E ⁰ = -1.18 V (based on ligand reduction) ), Its selective electrocatalytic reduction of carbon dioxide to carbon monoxide (Reference 27). In order to promote efficient electron transfer between the PSP and the catalyst, the present inventors synthesized N- (2,6,2-terpyridin-4-yl) -iodoacetamide (Compound 7, Example 1). By introducing cysteine mutations at different sites of PSP2, N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) was specifically conjugated to PSP2 (to obtain LC- The MS spectrum confirms that, see Figure 10, Compound 7 is specifically conjugated to the introduced cysteine). Then, the inventors determined the efficiency of these cysteine mutants to catalyze a photocatalytic carbon dioxide reduction reaction in the presence of divalent nickel ions. Add Ni (ClO ₄ ) ₂ , NaHCO ₃ and 4- (2,3-dihydro-1H-benzo [d] imidazol-2-yl) benzene-1,2-diol to the modified PSP2 mutant protein (BIH) as a sacrificial reducing agent. This quantitatively formed a PSP2-terpyridine nickel (II) complex (FIG. 11), and the sample was irradiated with a solar simulator (wavelength λ> 400 nm) for 12 hours. As shown in Figure 3b, although the PSP2-147C mutant exhibited low carbon monoxide production activity (number of conversions (TON) = 11), as the catalyst / chromophore distance changed from

Increase to

(Figure 12), the catalytic activity increased, reaching the highest level in PSP2-95C (TON = 75). However, a further increase in catalyst / chromophore distance resulted in a reduction in carbon monoxide production (Figure 3b). In all cases, no H ₂ or HCOOH was detected. These results indicate that effective photocatalytic carbon dioxide reduction requires an optimal catalyst / chromophore distance. Because the PSP2-95C mutant has a higher carbon dioxide reducing activity after being modified with a terpyridine nickel (II) complex, it is called PSP2T1. Conditions in the absence of Ni (II), NaHCO ₃ , BIH, BpA66 incorporation or PSP and N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) Below, no significant amount of carbon monoxide is generated, which indicates that all the above components are necessary for photocatalytic carbon dioxide reduction (Figure 3c). To verify the formation of heterogeneous particles during photolysis, transmission electron microscopy (TEM) experiments were performed on samples before and after photolysis. As shown in Figure 13, the TEM photos of the samples before and after photolysis are similar, indicating that no heterogeneous particles were generated during photolysis. These results confirm that PSP2 · produced by photochemistry can reduce the nickel-terpyridine complex (E ⁰ = -1.0V), and therefore PSP2 · has a reduction potential lower than -1.0V.

In order to study whether the presence of a local proton donor improves the catalytic efficiency ¹² , the inventors designed and produced a mutant PSP2-95C93Y97Y (SEQ ID NO: 10). The inventors envisage that the presence of two tyrosine residues at 95C covalently linked to the catalyst terpyridine nickel complex may serve as a local proton donor to promote proton-coupled electron transfer to the carbon dioxide substrate, which would reduce the energy for carbon dioxide reduction Obstacle ⁵ . In fact, PSP2-95C93Y97Y was modified by the catalyst N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) and behaved in the presence of divalent nickel ions (ie, PSP2T2 was obtained). Significantly increased TON (Figure 3c / d). The inventors have calculated that PSP2T2 has a quantum yield of 2.6% for photocatalytic reduction of carbon dioxide to carbon monoxide (Table 2-3). In contrast, using CdS nanorods as the photosensitizer and using the same nickel-terpyridine catalyst, only a 0.28% CO ₂ / CO conversion quantum yield of ^{27 was} observed. Here, compared with the photo-excited state of CdS, the reduction potential of PSP2 · is much lower, and the ability to accurately control the chromophore / catalyst distance and optimization of the catalyst microenvironment lead to a nine-fold improvement in efficiency.

In order to study the PSP mechanism, the inventors measured the transient absorption spectrum of the transient species produced by photoexcitation. When PSP2 was irradiated with a 355 nm laser, the inventors observed a recovery of the absorption at the 380 nm ground state and the attenuation of a new peak at 430 nm, which indicates the formation of the PSP2 triplet excited state (PSP2 *, Figure 4a / b). The decay lifetime of PSP2 * is 123 μs, which is about 10 ⁵ times the lifetime of the singlet excited state of fluorescent proteins, and is such as tris (bipyridine) ruthenium (II) halide ([Ru (bpy) ₃ ] ²⁺ ) (~ 0.5 μs ) The triplet excited state lifetime of the photosensitizer is more than 200 times ²⁸ . The long lifetime of PSP2 * is important to promote an efficient electron transfer reaction between the electron donor and PSP2. The transient absorption spectrum shows that as the ascorbate concentration increases, the PSP2 * lifetime decreases and a new peak appears at 500-560nm. These results indicate that PSP2 * reacts with ascorbate to produce PSP2 · with a secondary rate constant of 2.2 × 10 ⁵ M ^-1 s ^-1 (Figure 4c / d). If the catalyst terpyridine nickel complex is too close to the chromophore, both forward electron transfer and charge recombination occur rapidly, which prevents the formation of the 2e reduced state of the terpyridine nickel complex. If the terpyridine nickel complex is far from the chromophore, the electron transfer from PSP2 * to the terpyridine nickel complex is too slow to support efficient carbon dioxide reduction. The distance between the catalyst and the chromophore must be exactly (Table 3), so that after PSP2 * is reduced by the sacrificial reducing agent, the catalyst is reduced by one electron, and the second electron transfer competitive inhibition from PSP2 · to the terpyridine nickel complex Charge recombination (Figure 4e / f). Here, the advantages of using tiny proteins as self-assembled photocatalytic units are clearly visible: the catalyst / chromophore distance and their microenvironment can be accurately and conveniently designed by directional mutagenesis, thereby optimizing catalyst performance.

Table 2. PSP2T1 and PSP2T2 conversion number (TONs) and quantum efficiency (QE) data

Table 3. Reaction conditions and evaluation catalysts N- (2,6,2-terpyridin-4-yl) -iodoacetamide (compound 7) modified various PSP2 monocysteine mutants on divalent nickel ions The results obtained in the presence of the catalytic performance are summarized.

C represents cysteine, and the number before C indicates the site of mutation to cysteine.

in conclusion

In summary, by utilizing the genetic codon extension ¹⁵ , the inventors reasonably designed an effective photosensitizing protein PSP2, which can be regarded as a protein analogue of [Ru (bpy) ₃ ] ²⁺ containing no precious metals. Since PSP2 is genetically encoded, it can be easily introduced into various organisms and co-localized with specific protein complexes. This is difficult to achieve with small molecule or nanocrystalline photosensitizers. Compared with semiconductor nanocrystals and small molecule photosensitizers, PSP offers unique advantages, such as higher compatibility with a wide range of biological systems, no dependence on precious metals, mutation-based switchable photochemical properties, and self-assembly The ability to create accurate three-dimensional structures, which can allow for modular extension of its functionality and accurate mechanism characterization. As a result, PSP can potentially sensitize a variety of challenging chemical transformations, involving diverse fields such as solar energy conversion, photobiology, environmental restoration, and industrial biology. PSP2T simple design captures the essence of ^3-4 complex natural photosynthesis mechanism provides a valuable model for the study of protein ²⁹ in a variety of electronic mechanisms / proton transfer, and through rational design and directed evolution for the next generation The foundation of photoreductases with significantly expanded capabilities is laid. Photosensitizers, such as the most famous [Ru (bpy) ₃ ] ²⁺ , have led the revolution in synthetic chemistry ²⁸ . Utilizing the extraordinary self-assembly capabilities of proteins ^30-31 , diverse enzyme-catalyzed reactions, and the inventor's ability to rapidly improve the rational design of miniature proteins ^32-40 , the design of PSP will introduce new chemical reactions in biological systems Created multiple exciting opportunities.

It should be understood that although the present invention has been specifically shown and described with reference to its exemplary embodiments, those of ordinary skill in the art will understand that without departing from the spirit and scope of the present invention as defined by the appended items Under the conditions, various forms and details can be changed therein, and any combination of various embodiments can be made.

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Claims (25)

1. An artificial photosynthesis protein comprising a fluorescent protein (FP) in which a chromophore amino acid residue is modified by a photosensitizer to be converted into a photosensitive protein (PSP).
2. The artificial photosynthetic protein according to claim 1, wherein the fluorescent protein comprises a dark blue fluorescent protein such as Sirius, a blue fluorescent protein such as EBFP, Azurite, EBFP2, TagBFP, a cyan fluorescent protein such as ECFP, Cerulean, CyPet, mTurquoise, mTFP1 (Teal), Midoriishi-Cyan, green fluorescent proteins such as GFP, UKG, EGFP, Emerald, Superfolder, yellow fluorescent proteins such as YFP, sfYFP, EYFP, Venus, Citrine, YPet, PhiYFP, orange fluorescent proteins such as mHoneydew, mBanana, mKO, mKOκ, mOrange, mOrange2, red fluorescent proteins such as TagRFP, TagRFP158T, mRuby, mCherry, deep red fluorescent proteins such as Katushka, mKate, mKate2, mPlum, E2-Crimson, mNeptune, Tag657, eqFP650, eqFP670, near-infrared fluorescent proteins such as mIFP , Bacterial light-sensitive pigment proteins such as BphP, phycobiliproteins such as phycoerythrin CPE, phycocyanin PEC, phycocyanin CPC, phycocyanin APC, or variants thereof.
3. The artificial photosynthetic protein according to claim 1 or 2, wherein the photosensitizer is a photosensitizer encoded by an extended genetic code, such as benzophenone-alanine BpA.
4. The artificial photosynthetic protein according to any one of claims 1 to 3, wherein the chromophore amino acid residues include, for example, amino acid residues corresponding to positions 65, 66, and 67 of the green fluorescent protein GFP, particularly corresponding to green Tyrosine residue at position 66 of the fluorescent protein GFP.
5. The artificial photosynthetic protein according to any one of claims 1 to 4, wherein the modification includes introducing the photosensitizer by modifying, inserting, replacing, or deleting amino acid residues the amino acid sequence encoding a wild-type fluorescent protein, such as extending the genetic code Encoded photosensitizer, such as benzophenone-alanine BpA.
6. The artificial photosynthetic protein according to any one of claims 1 to 5, further comprising modification of amino acid residues other than chromophore residues, such as amino acid residue insertions, substitutions, and deletions.
7. The artificial photosynthetic protein according to any one of claims 1-6, wherein the modification of the amino acid residue includes a modification capable of obtaining one or more of the following properties: 1) adjusting the photochemical characteristics of the chromophore, such as making it photoactive The excited state is sufficiently oxidizing, capable of oxidizing the weak sacrificial reducing agent, and generating a strong reducing group that promotes the reduction of the carbon dioxide reduction catalyst; 2) adjusting the distance between the chromophore and the catalytic center, and promoting the transition from the chromophore to the catalytic center Continuous electron transfer steps to prevent unfavorable charge recombination; and / or 3) adjust the microenvironment of the catalytic center to optimize the transfer of protons and electrons.
8. The artificial photosynthetic protein according to any one of claims 1 to 7, which is an artificial photosynthetic protein having one or more of the following properties: (1) capable of effectively absorbing visible light; (2) capable of absorbing photons, Transform into a long-existing photo-excited state (PSP *), thereby promoting the electron transfer reaction, leading to the formation of PSP radicals (PSP ·); and / or (3) PSP · is a strong reducing agent that can drive the reduction of the carbon dioxide reduction catalyst .
9. The artificial photosynthetic protein according to any one of claims 1 to 8, comprising

   a) the amino acid sequence shown in any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,

   b) has a sequence shown in any of the above with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher Amino acid sequence of amino acid identity;

   c) has one or more sequences (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16 17, 18, 19, 20, 21, 22, 23, 24, 25 or more, preferably one or several, such as 1, 2, 3, 4, 5 or more) amino acid sequences with amino acid differences.
10. A photosensitive carbon dioxide reductase, comprising the artificial photosynthetic protein according to any one of claims 1 to 9 and a carbon dioxide reduction catalyst conjugated to the artificial photosynthetic protein.
11. The photosensitive carbon dioxide reductase of claim 10, wherein the carbon dioxide reduction catalyst comprises a terpyridine nickel (II) complex.
12. The photosensitive carbon dioxide reductase according to claim 10 or 11, wherein the carbon dioxide reduction catalyst is directly conjugated to an amino acid residue of the artificial photosynthesis protein, or indirectly conjugated to the artificial photosynthesis protein through a linker. Amino acid residues.
13. The photosensitive carbon dioxide reductase of claim 12, wherein the amino acid residue conjugated to the carbon dioxide reduction catalyst includes an amino acid residue introduced by modification of an amino acid residue.
14. The photosensitive carbon dioxide reductase according to any one of claims 10 to 13, wherein the carbon dioxide reduction catalyst is directly or indirectly conjugated to a cysteine residue, such as an cysteine residue introduced by modifying an amino acid residue.
15. The photosensitive carbon dioxide reductase according to any one of claims 10 to 14, which is free of precious metals and / or independent of precious metals.
16. An artificial photosynthesis system comprising the artificial photosynthesis protein according to any one of claims 1-9 or the photosensitive carbon dioxide reductase according to any one of claims 10-15, and preferably the artificial photosynthesis system is Gene-coded artificial photosynthesis system.
17. A method for preparing an artificial photosynthetic protein, comprising: modifying a chromophore amino acid residue of a fluorescent protein (FP) with a photosensitizer, thereby converting it into a photosensitized protein (PSP).
18. The method of claim 17, further comprising modification of amino acid residues other than chromophore residues, such as amino acid residue insertions, substitutions, and deletions, for example, by substitution of charged residues to regulate PSP chromophore reduction Potential.
19. The method of claim 17 or 18, comprising detecting a color change of the modified product to determine the occurrence of a photochemical reaction.
20. The method according to any one of claims 17 to 19, comprising detecting the reducing ability of the modified product, such as a photochemical reduction of a weak reducing agent such as ascorbic acid.
21. The method according to any one of claims 17 to 19, comprising 1) performing UV-Vis titration, such as UV-Vis titration under different pH conditions; 2) collecting X-band electron spin resonance data; 3) performing Electrochemical detection of proteins; 4) detection of reactivity with oxygen; and / or 5) determination of crystal structure, such as determination of crystal structure by X-ray diffraction.
22. A fusion protein comprising the artificial photosynthesis protein according to any one of 1-9.
23. A nucleic acid molecule encoding the artificial photosynthesis protein according to any one of claims 1-9 or the fusion protein according to claim 22, preferably the codons of the nucleic acid molecule are optimized to be suitable for corresponding host cells Expression, for example, the codons of the nucleic acid molecule are optimized for expression in mammalian cells.
24. A recombinant expression system comprising the nucleic acid molecule according to claim 23.
25. A recombinant host cell comprising the expression system of claim 24.

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