Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha‐helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis₃ or CuHis₃ site, or that recapitulate the spectroscopic properties of unique electron‐transfer sites in cupredoxins (CuHis₂Cys) or rubredoxins (FeCys₄). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000‐fold.     

Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acid

While nature employs various covalent and non-covalent strategies to modulate tyrosine (Y) redox potential and pK _a in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Through the genetic incorporation of 3-methoxytyrosine (OMeY) into myoglobin, we replicated important features of cytochrome c oxidase (CcO) in this small soluble protein, which exhibits selective O₂ reduction activity while generating a small amount of reactive oxygen species (ROS). These results demonstrate that the electron donating ability of a tyrosine residue in the active site is important for CcO function. Moreover, we elucidated the structural basis for the genetic incorporation of OMeY into proteins by solving the X-ray structure of OMeY specific aminoacyl-tRNA synthetase complexed with OMeY.     

Rational Heme Protein Design: All Roads Lead to Rome

Heme proteins are among the most abundant and important metalloproteins, exerting diverse biological functions including oxygen transport, small molecule sensing, selective C? H bond activation, nitrite reduction, and electron transfer. Rational heme protein designs focus on the modification of the heme‐binding active site and the heme group, protein hybridization and domain swapping, and de novo design. These strategies not only provide us with unique advantages for illustrating the structure–property–reactivity–function (SPRF) relationship of heme proteins in nature but also endow us with the ability to create novel biocatalysts and biosensors.     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.     

Metalloproteins/metalloenzymes for the synthesis of acetyl-CoA in the Wood-Ljungdahl pathway

This paper focuses on the group of metalloproteins/metalloenzymes in the acetyl-coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway, including formate dehydrogenase (FDH), corrinoid iron sulfur protein (CoFeSP), acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH, a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate, catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP, as a methyl group transformer, accepts the methyl group from CH₃-H₄ folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO₂ to CO and ACS functions for acetyl-coenzyme A synthesis through condensation of the methyl group, CO and coenzyme A, to finish the whole pathway. This paper introduces the structure, function and reaction mechanisms of these enzymes.     

Repeat Module‐Based Rational Design of a Photoswitchable Protein for Light‐Driven Control of Biological Processes

Light‐driven control of biological processes using photoswitchable proteins allows high spatiotemporal interrogation or manipulation of such processes, assisting in understanding their functions. Despite considerable advances, however, the wide spread use of optical control has been hampered by a limited repertoire of photoswitchable proteins and a lack of generalized design strategy. Herein, we present a repeat module‐based rational design of a photoswitchable protein composed of LRR (Leucine‐rich repeat) modules using azobenzene as a photochromic ligand. Our design approach involves the rational selection of a C_β pair between two nearby modules within a convex region and subsequent cross‐linking with a photochromic ligand. We demonstrate the general utility and potential of our strategy by showing the design of three target‐specific photoswitchable proteins and a light‐driven modulation of the cell signaling. With an abundance of LRR proteins in nature, our approach can expand the repertoire of photoswitchable proteins for light‐driven control of biological processes.     

Reversible Unfolding–Refolding of Rubredoxin: A Single‐Molecule Force Spectroscopy Study

In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding–refolding of the iron–sulfur protein rubredoxin was observed directly using single‐molecule force spectroscopy. The results demonstrate that the iron can remain attached to the CXXC motif when rubredoxin is unfolded. Upon relaxation, the unfolded rubredoxin can refold into its native holo state with the reconstituted FeS₄ center. The possible loss of iron from the unfolded protein prevents rubredoxin from refolding into its native holo state. These results demonstrated that unfolding of rubredoxin is reversible, as long as the iron remains attached, and provide experimental evidence for the iron‐priming mechanism for the folding of rubredoxin.     

计算机辅助蛋白质分子理性设计:从肌红蛋白到一氧化氮还原酶

计算机辅助蛋白质分子理性设计在解决化学及生物学重要问题中被证实十分有效。在NOR本身三维结构未知的情况下通过计算机分子模拟,使用肌红蛋白(Mb)作为蛋白质分子模型,设计了结构功能型一氧化氮还原酶(NOR),所设计的NOR蛋白质模型——Fe_BMb一年后被天然NOR的晶体结构所证实。本文综述了设计Fe_BMb, I107E Fe_BMb以及Fe_BMb(-His)的研究过程及其设计合理性,评述了通过使用计算机分子模拟,获得Mb处于双组氨酸配位的非天然状态的原子层次结构信息,而这些信息很难通过实验方法来获得。计算机辅助蛋白质分子理性设计的广泛应用将会为生物体系提供更深刻的内涵。     

细胞色素b5-蛋白质相互作用研究

蛋白质-蛋白质相互作用在生命过程中发挥至关重要的作用,特别是血红素类蛋白。细胞色素b5(Cyt b5)是血红素蛋白的一个典型代表,在生物体内通过多种蛋白质-蛋白质相互作用来执行其生物功能。目前所揭示的与Cyt b5相关的蛋白质相互作用包括:细胞色素b5-细胞色素b5还原酶,细胞色素b5-细胞色素P450,细胞色素b5-细胞色素c,细胞色素b5-肌红蛋白或血红蛋白,细胞色素b5-融合蛋白(谷胱甘肽S-转移酶GST和绿色荧光蛋白GFP)和细胞色素b5-转运蛋白(蔗糖转运蛋白SUT1和山梨醇转运蛋白SOT6)等。同一蛋白能与众多不同蛋白相互作用的事实,使我们认识到某些特定蛋白的生物学重要性。另一方面,研究同一蛋白与不同蛋白质间的相互作用将会进一步加深我们对蛋白质结构与功能关系的理解,以及指导新颖蛋白的理性设计与最终应用。     

D-A type (dfppy)2Ir(pic-TPA) complex containing fluorinated pyridine-2-carboxylate ligand and triphenylamine: synthesis,photophysics and bioactivity

A novel Ir (III) complex (dfppy) ₂ Ir(pic-TPA) containing fluorinated phenylpyridine and pyridine-2-carboxylate ancillary ligand was synthesized according to rational design, its photophysical properties and therapeutical activities examined. Radiosensitization SER values (1.63-1.65) were observed even in radio-resistant cell lines after treatment with this complex. Furthermore, the complexes have shown antiviral activity against HSV-2 DNA virus. Comply with sub-cellular distribution imaging results and molecular docking calculations and cell cytometry analysis, this Ir (III) complex demonstrated functions as promising microviscosiy and micropolarity imaging probe, also as a prodrug for combinatorial therapy of radiochemo and antiviral treatment.     

An object-oriented library for computational protein design

Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.     

XAFS study of Ni (II)–aminovinylketone complexes

The functional properties of the active sites in a metalloproteins depend on coordination geometry of metal, the number and the nature of coordination ligands. The Ni K-edge XAFS spectra of novel nickel complexes as models for the MeN₂O₂(S₂) active site in metalloproteins were measured and analyzed. Theoretical analysis of the Ni K-edge XANES was performed using FDMNES code based on the finite difference method (FDM) to solve the Schrödinger equation beyond muffin-tin approximations and self-consistent full multiple-scattering approach (code FEFF8.2). It was found that the spectrum is almost totally formed by the octahedron of the nearest neighbor atoms around Ni ion in the II (Ni) complex. The III (Ni) complex active center exists in square-planar configuration with shorter distances.     

An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1

Metal clusters, such as iron–sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins. Herein we report the formation and crystal structure of a planar square tetranuclear silver cluster when silver ions were mixed with human copper chaperone Atox1. Quantum chemical studies reveal that two Ag 5s¹ electrons in the tetranuclear silver cluster fully occupy the one bonding molecular orbital, with the assumption that this Ag₄ cluster is Ag₄²⁺, leading to extensive electron delocalization over the planar square and significant stabilization. This bonding pattern of the tetranuclear silver cluster represents an aromatic all-metal structure that follows a 4n + 2 electron counting rule (n = 0). This is the first time an all-metal aromatic silver cluster was observed in a protein.

Metal clusters, such as iron–sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins.     

Protein dynamics and function: Making new strides with an old warhorse,the alkaline conformational transition of cytochrome c

Protein dynamics is intimately linked to function. In metalloproteins, dynamics are often coupled to redox activity, ligand binding and enzyme function. We provide a concise overview of the field and then focus on the use of the alkaline conformer of cytochrome c as a model system to probe the factors that control the conformational dynamics of proteins in general and metalloproteins in particular. We consider the effects of ligands on metal-mediated dynamics, the interplay between intrinsic metal-ligand dynamics and barriers imposed by the protein scaffold itself, and the effects of local and overall protein stability on dynamics. Discussed within are the collected results from equilibrium thermodynamic methods, pH jump kinetics and conformationally gated redox reactions between small inorganic reagents and metalloproteins used as a means to probe conformational switching in metalloproteins.     

Nanoconfinement-Guided Construction of Nanozymes for Determining H2O2 Produced by Sonication

Nanomaterials with enzyme-like activities, termed as nanozymes, have found wide applications in various fields. It has been a long-term aim to rationally design and synthesize highly active nanozymes and thus to further improve their application performance. Guided by the nanoconfinement effect, we confine cytochrome c (Cyt c) within a mesoporous metal–organic framework (MOF), PCN-222 nanoparticle (NP), forming a protein/MOF hybrid nanozyme, termed as Cyt c@PCN-222 NP. The confined Cyt c exhibits around 3–4-fold higher peroxidase-like activity than free Cyt c. Due to the increase in the activity of Cyt c, the Cyt c@PCN-222 NPs exhibit a quite low limit of detection (≈0.13 μM) towards H₂O₂. Sonication-induced H₂O₂ formation in water by using a lab-quipped ultrasonic cleaner can be sensitively probed, which suggests that H₂O₂-sensitive materials should be carefully handled during the utilization of ultrasonic equipment. We speculate that this nanoconfinement strategy can broaden our synthetic methodology for the rational design of nanozymes.     

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a₃ in caa₃-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN⁻ ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.     

Altering Oxygen Binding by Redox-Inactive Metal Substitution to Control Catalytic Activity: Oxygen Reduction on Manganese Oxide Nanoparticles as a Model System**

Establishing generic catalyst design principles by identifying structural features of materials that influence their performance will advance the rational engineering of new catalytic materials. In this study, by investigating metal-substituted manganese oxide (spinel) nanoparticles, Mn₃O₄:M (M=Sr, Ca, Mg, Zn, Cu), we rationalize the dependence of the activity of Mn₃O₄:M for the electrocatalytic oxygen reduction reaction (ORR) on the enthalpy of formation of the binary MO oxide, Δ_fH°(MO), and the Lewis acidity of the M²⁺ substituent. Incorporation of elements M with low Δ_fH°(MO) enhances the oxygen binding strength in Mn₃O₄:M, which affects its activity in ORR due to the established correlation between ORR activity and the binding energy of *O/*OH/*OOH species. Our work provides a perspective on the design of new compositions for oxygen electrocatalysis relying on the rational substitution/doping by redox-inactive elements.     

Design, folding, and activities of metal-assembled coiled coil proteins

Metal ions serve many purposes in natural proteins, from the stabilization of tertiary structure to the direction of protein folding to crucial roles in electron transfer and catalysis. There is considerable interest in creating metal binding sites in designed proteins to understand the structural role of metal ions and to design new metalloproteins with useful functions. The de novo design of metalloproteins and the role of metals in the folding of designed proteins are reviewed here, with particular focus on the design, folding, and activities of the [M(bpy-peptide)(3)](2+) structure. This maquette is constructed by the covalent attachment of 2,2'-bipyridine to the N-termini of amphiphilic peptides, and it is assembled into a folded trimeric coiled coil by the addition of a six-coordinate transition metal ion and the resulting hydrophobic collapse of the peptides. The [M(bpy-peptide)(3)](2+) structure has been employed in diverse applications, ranging from electron transfer pathway studies to the study of optimal hydrophobic packing in a virtual library to the construction of receptors and biosensors.     

Use of proteomics for design of a tailored host cell for highly efficient protein purification

After some initial optimization, a downstream process comprised of one or several chromatography steps removes the majority of the host proteins and achieves a reasonable degree of purification. The separation of remaining contaminant proteins from the target protein could become very difficult and costly due to their similar physicochemical properties. In this paper we describe a highly efficient strategy, based on proteomic analysis and elution chromatography, by which a protein of interest may be isolated from copurifying contaminants. Mutant strains of Escherichia coli were prepared that are deficient in three prevalent host proteins found in a strategic fraction of an elution profile of nickel immobilized affinity chromatography. Recombinant green fluorescent protein (GFPuv) served as a model protein and its elution was directed to this optimized fraction with an N-terminus hexahistidine tag (his₆), thereby easing its recovery. We demonstrate that proteomic data can facilitate the rational engineering of host cell expressing the target protein and the design of an efficient process for its purification.     

Oxidation of organic molecules in homogeneous aqueous solution catalyzed by hybrid biocatalysts (based on the Trojan Horse strategy)

New anionic metalloporphyrin–estradiol conjugates have been synthesized and fully characterized, and have been further associated to a monoclonal anti-estradiol antibody 7A3, to generate new artificial metalloenzymes following the so-called ‘Trojan Horse’ strategy. The spectroscopic characteristics and dissociation constants of these complexes were similar to those obtained for the artificial metalloproteins obtained by association of cationic metalloporphyrin–estradiol conjugates to 7A3. This demonstrates that the nature of the porphyrin substituents, anionic or cationic, had little influence on the association with the antibody that is mainly driven by the tight association of the estradiol anchor with the binding pocket of the antibody.These new biocatalysts appeared to have an interesting catalytic activity in oxidation reactions. The iron(III)–anionic-porphyrin–estradiol-antibody complexes were found able to catalyze the chemoselective and slightly enantioselective (ee = 10%) sulfoxidation of sulfides by H₂O₂. The Mn(III)–porphyrin–estradiol-antibody complexes were found to catalyze the oxidation of styrene by KHSO₅, the Mn(III)–cationic-porphyrin–estradiol-antibody complexes even showing the highest yields so far reported for the oxidation of styrene catalyzed by artificial metalloproteins. However, a lack of chemoselectivity and enantioselectivity was observed, which was probably due to a weak interaction of the metalloporphyrin cofactor with the binding pocket of antibody 7A3, as suggested by the similar UV–visible characteristics and catalytic activities obtained with both anionic and cationic porphyrins.