On the hunt for metalloenzyme inhibitors: Investigating the presence of metal-coordinating compounds in screening libraries and chemical spaces.

Metalloenzymes play vital roles in various biological processes, requiring the search for inhibitors to develop treatment options for diverse diseases. While compound library screening is a conventional approach, the exploration of virtual chemical spaces housing trillions of compounds has emerged as an alternative strategy. In this study, we investigated the suitability of selected screening libraries and chemical spaces for discovering inhibitors of metalloenzymes featuring common ions (Mg2+, Mn2+, and Zn2+). First, metal-coordinating groups from ligands interacting with ions in the Protein Data Bank were extracted. Subsequently, the prevalence of these groups in two focused screening libraries (Life Chemicals' chelator library, comprising 6,428 compounds, and Otava's chelator fragment library, with 1,784 fragments) as well as two chemical spaces (GalaXi and REAL space, containing billions of virtual products) was investigated. In total, 1,223 metal-coordinating groups were identified, with about a quarter of these groups found within the examined libraries and spaces. Our results indicate that these can serve as valuable starting points for drug discovery targeting metalloenzymes. In addition, this study suggests ways to improve libraries and spaces for better success in finding potential inhibitors for metalloenzymes.

Development of a Fluctuating Charge Model for Zinc-Containing Metalloproteins.

Metalloproteins widely exist in biology and play important roles in various processes. To accurately simulate metalloprotein systems, modeling polarization and charge transfer effects is vital. The fluctuating charge (FQ) model can efficiently generate atomic charges and simulate the charge transfer effect; it has been developed for a wide range of applications, but few models have been specifically tailored for metalloproteins. In this study, we present a fluctuating charge model specifically for zinc-containing metalloproteins based on the extended charge equilibration (EQeq) scheme. Our model was parametrized to reproduce CM5 charges instead of RESP/CHELPG charges because the former is less dependent on the conformation or basis set, does not suffer from unphysical charges for buried atoms, and is still being able to well reproduce the molecular dipoles. During our study, we found that adding the Pauling-bond-order-like term (referred to as the "+C term" in a previous study) between the zinc ion and ligating atoms significantly improves the model's performance. Although our model was trained for four-coordinated zinc sites only, our results indicated it can well describe the atomic charges in diverse zinc sites. Morever, our model was used to generate partial charges for the metal sites in three different zinc-containing metalloproteins (with four-, five-, and six-coordinated metal sites, respectively). These charges exhibited performance comparable to that of the RESP charges in molecular dynamics (MD) simulations. Additional tests indicated our model could also well reproduce the CM5 charges when geometric changes were involved. Those results indicate that our model can efficiently calculate the atomic charges for metal sites and well simulate the charge transfer effect, which marks an important step toward developing versatile polarizable force fields for metalloproteins.

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography.

Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

The study of acidic/basic nature of metallothioneins and other metal-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius).

Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4-6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ.

Acclimation to medium-level non-lethal iron limitation: Adjustment of electron flow around the PSII and metalloprotein expression in Trichodesmium erythraeum IMS101.

The aim of this study was to investigate how acclimation to medium-level, long-term, non-lethal iron limitation changes the electron flux around the Photosystem II of the oceanic diazotroph Trichodesmium erythraeum IMS101. Fe availability of about 5× and 100× lower than a replete level, i.e. conditions common in the natural environment of this cyanobacterium, were applied in chemostats. The response of the cells was studied not only in terms of growth, but also mechanistically, measuring the chlorophyll fluorescence of dark-adapted filaments via imaging fluorescence kinetic microscopy (FKM) with 0.3 ms time resolution. Combining these measurements with those of metal binding to proteins via online coupling of metal-free HPLC (size exclusion chromatography SEC) to sector-field ICP-MS allowed to track the fate of the photosystems, together with other metalloproteins. General increase of fluorescence has been observed, with the consequent decrease in the quantum yields φ of the PSII, while the efficiency ψ of the electron flux between PSII and the PSI remained surprisingly unchanged. This indicates the ability of Trichodesmium to cope with a situation that makes assembling the many iron clusters in Photosystem I a particular challenge, as shown by decreasing ratios of Fe to Mg in these proteins. The negative effect of Fe limitation on PSII may also be due to its fast turnover. A broader view was obtained from metalloproteomics via HPLC-ICP-MS, revealing a differential protein expression pattern under iron limitation with a drastic down-regulation especially of iron-containing proteins and some increase in low MW metal-binding complexes.

Coordination chemistry of mitochondrial copper metalloenzymes: exploring implications for copper dyshomeostasis in cell death.

Mitochondria, fundamental cellular organelles that govern energy metabolism, hold a pivotal role in cellular vitality. While consuming dioxygen to produce adenosine triphosphate (ATP), the electron transfer process within mitochondria can engender the formation of reactive oxygen species that exert dual roles in endothelial homeostatic signaling and oxidative stress. In the context of the intricate electron transfer process, several metal ions that include copper, iron, zinc, and manganese serve as crucial cofactors in mitochondrial metalloenzymes to mediate the synthesis of ATP and antioxidant defense. In this mini review, we provide a comprehensive understanding of the coordination chemistry of mitochondrial cuproenzymes. In detail, cytochrome c oxidase (CcO) reduces dioxygen to water coupled with proton pumping to generate an electrochemical gradient, while superoxide dismutase 1 (SOD1) functions in detoxifying superoxide into hydrogen peroxide. With an emphasis on the catalytic reactions of the copper metalloenzymes and insights into their ligand environment, we also outline the metalation process of these enzymes throughout the copper trafficking system. The impairment of copper homeostasis can trigger mitochondrial dysfunction, and potentially lead to the development of copper-related disorders. We describe the current knowledge regarding copper-mediated toxicity mechanisms, thereby shedding light on prospective therapeutic strategies for pathologies intertwined with copper dyshomeostasis. [BMB Reports 2023; 56(11): 575-583].

Be2+ Causes Hypersensitivity but Mg2+ and Ca2+ Do Not─Favorable Metal Coordination Is the Key for Differential Allosteric Modulation and Binding Affinities.

Although the ion selectivity of metalloproteins has been well established, selective metal antigen recognition by immunoproteins remains elusive. One such case is the recognition of the Be2+ ion against its heavier congeners, Mg2+ and Ca2+, by the human leukocyte antigen immunoprotein (HLA-DP2), leading to immunotoxicity. Integrating with our previous mechanistic study on Be2+ toxicity, herein, we have explored the basis of characteristic nontoxicity of Mg2+ and Ca2+ ions despite their in vivo abundance. The ion binding cleft of the HLA-DP2-peptide complex is composed of four acidic residues, p4D and p7E from the peptide and ß26E and ß69E from the protein. While the tetrahedral coordination site of the smaller Be2+ ion is located deep inside the cavity, hexa- to octa-coordination sites of Mg2+ and Ca2+ ions are located closer to the protein surface. The intrinsic high coordination number of Mg2+/Ca2+ ions induces allosteric modifications on the HLA-DP2_M2 surface, which are atypical for TCR recognition. Furthermore, the lower binding energy of larger Mg2+ and Ca2+ ions with the cavity residues can be correlated to the lower charge density and reduced covalent bonding nature as compared to those of the smaller Be2+ ion. In short, weak binding of Mg2+ and Ca2+ ions and the unfavorable allosteric surface modifications are probably the major determinants for the absence of Mg2+/Ca2+ ion-mediated hypersensitivity in humans.

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in Escherichia coli.

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe8S7]) and M-cluster core (or L-cluster; [Fe8S9C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

TerC proteins function during protein secretion to metalate exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

Chemoproteomics Reveals Disruption of Metal Homeostasis and Metalloproteins by the Antibiotic Holomycin.

The natural product holomycin contains a unique cyclic ene-disulfide and exhibits broad-spectrum antimicrobial activities. Reduced holomycin chelates metal ions with a high affinity and disrupts metal homeostasis in the cell. To identify cellular metalloproteins inhibited by holomycin, reactive-cysteine profiling was performed using isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). This chemoproteomic analysis demonstrated that holomycin treatment increases the reactivity of metal-coordinating cysteine residues in several zinc-dependent and iron-sulfur cluster-dependent enzymes, including carbonic anhydrase II and fumarase A. We validated that holomycin inhibits fumarase A activity in bacterial cells and diminishes the presence of iron-sulfur clusters in fumarase A. Whole-proteome abundance analysis revealed that holomycin treatment induces zinc and iron starvation and cellular stress. This study suggests that holomycin inhibits bacterial growth by impairing the functions of multiple metalloenzymes and sets the stage for investigating the impact of metal-binding molecules on metalloproteomes by using chemoproteomics.

Metalloproteome of human-infective RNA viruses: a study towards understanding the role of metal ions in virology.

Metalloproteins and metal-based inhibitors have been shown to effectively combat infectious diseases, particularly those caused by RNA viruses. In this study, a diverse set of bioinformatics methods was employed to identify metal-binding proteins of human RNA viruses. Seventy-three viral proteins with a high probability of being metal-binding proteins were identified. These proteins included 40 zinc-, 47 magnesium- and 14 manganese-binding proteins belonging to 29 viral species and eight significant viral families, including Coronaviridae, Flaviviridae and Retroviridae. Further functional characterization has revealed that these proteins play a critical role in several viral processes, including viral replication, fusion and host viral entry. They fall under the essential categories of viral proteins, including polymerase and protease enzymes. Magnesium ion is abundantly predicted to interact with these viral enzymes, followed by zinc. In addition, this study also examined the evolutionary aspects of predicted viral metalloproteins, offering essential insights into the metal utilization patterns among different viral species. The analysis indicates that the metal utilization patterns are conserved within the functional classes of the proteins. In conclusion, the findings of this study provide significant knowledge on viral metalloproteins that can serve as a valuable foundation for future research in this area.

Cell-Free Protein Synthesis of Metalloproteins.

Metalloproteins, proteins containing metal atoms or clusters within their structures, are critical for various biological functions across all domains of life. More than hundreds of different types have been discovered, which conduct various roles such as transportation of O2, catalyzing chemical reactions, sensing environmental changes, and relaying electrons. Metalloprotein molecules incorporate a variety of metal atoms, coordinated to specific amino acid residues that affect their conformation and functionality. The process of metal incorporation typically occurs during or post-protein folding, often requiring chaperones for metal ion delivery and quality control. Progress in understanding metal incorporation and metalloprotein functionality has been enhanced by cell-free protein synthesis (CFPS) methods that offer direct control over the synthesis environment. This chapter reviews the diverse applications of CFPS methods in metalloprotein research, encompassing structure-function studies, protein engineering, and creation of artificial metalloproteins. Examples demonstrating the utility and advances brought about by CFPS in synthetic biology, electrochemistry, and drug discovery are highlighted. Despite remarkable progress, challenges remain in optimizing and advancing the CFPS methods, underscoring the need for future explorations in this transformative approach to metalloprotein study and engineering.

An Antibody Engineered Metalloenzyme for Mediating Cell-Cell Communication and Activation of Immuno- and Chemotherapy.

Artificial metalloenzymes (ArMs) are gaining much attention in life sciences. However, the function of the present ArMs for disease treatment is still in its infancy, which may impede the possible therapeutic potential. Herein, we construct an antibody engineered ArM by using the Fc region of IgG and bioorthogonal chemistry, which endows the ArM with the capability of manipulating cell-cell communication and bioorthogonal catalysis for tumor immuno- and chemotherapy. Specially, Fc-Pd ArM is modified on the cancer cell surface by metabolic glycoengineering to catalyze the bioorthogonal activation of prodrug for tumor chemotherapy. More importantly, the antibody-based ArM can mediate cell-cell communication between cancer cells and NK cells, activating the ADCC effect for immunotherapy. In vivo antitumor applications suggest that the ArM can not only eliminate primary tumor but also inhibit tumor lung metastasis. Our work provides a new attempt to develop artificial metalloenzymes with cell-cell communication the ability for bioorthogonal catalysis and combination therapy.

Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics.

Nitrite-driven anaerobic methane oxidation (N-damo) is a promising biological process to achieve carbon-neutral wastewater treatment solutions, aligned with the sustainable development goals. Here, the enzymatic activities in a membrane bioreactor highly enriched in N-damo bacteria operated at high nitrogen removal rates were investigated. Metaproteomic analyses, with a special focus on metalloenzymes, revealed the complete enzymatic route of N-damo including their unique nitric oxide dismutases. The relative protein abundance evidenced that "Ca. Methylomirabilis lanthanidiphila" was the predominant N-damo species, attributed to the induction of its lanthanide-binding methanol dehydrogenase in the presence of cerium. Metaproteomics also disclosed the activity of the accompanying taxa in denitrification, methylotrophy and methanotrophy. The most abundant functional metalloenzymes from this community require copper, iron, and cerium as cofactors which was correlated with the metal consumptions in the bioreactor. This study highlights the usefulness of metaproteomics for evaluating the enzymatic activities in engineering systems to optimize microbial management.

Oxidoreductases and metal cofactors in the functioning of the earth.

Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic-protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles.

Moving metals: How microbes deliver metal cofactors to metalloproteins.

First row d-block metal ions serve as vital cofactors for numerous essential enzymes and are therefore required nutrients for all forms of life. Despite this requirement, excess free transition metals are toxic. Free metal ions participate in the production of noxious reactive oxygen species and mis-metalate metalloproteins, rendering enzymes catalytically inactive. Thus, bacteria require systems to ensure metalloproteins are properly loaded with cognate metal ions to maintain protein function, while avoiding metal-mediated cellular toxicity. In this perspective we summarize the current mechanistic understanding of bacterial metallocenter maturation with specific emphasis on metallochaperones; a group of specialized proteins that both shield metal ions from inadvertent reactions and distribute them to cognate target metalloproteins. We highlight several recent advances in the field that have implicated new classes of proteins in the distribution of metal ions within bacterial proteins, while speculating on the future of the field of bacterial metallobiology.

Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

A De Novo Designed Trimeric Metalloprotein as a Nip Model of the Acetyl-CoA Synthase.

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.