The acetyl coenzyme A synthase (ACS) enzyme plays a central role in the metabolism of anaerobic bacteria and archaea, catalyzing the reversible synthesis of acetyl-CoA from CO and a methyl group through a series of nickel-based organometallic intermediates. Owing to the extreme complexity of the native enzyme systems, the mechanism by which this catalysis occurs remains poorly understood. In this work, we have developed a protein-based model for the Ni center of acetyl coenzyme A synthase using a nickel-substituted azurin protein (NiAz). NiAz is the first model nickel protein system capable of accessing three (Ni/Ni/Ni) distinct oxidation states within a physiological potential range in aqueous solution, a critical feature for achieving organometallic ACS activity, and binds CO and -CH groups with biologically relevant affinity. Characterization of the Ni-CO species through spectroscopic and computational techniques reveals fundamentally similar features between the model NiAz system and the native ACS enzyme, highlighting the potential for related reactivity in this model protein. This work provides insight into the enzymatic process, with implications toward engineering biological catalysts for organometallic processes.

Nickel-containing enzymes are key players in global hydrogen, carbon dioxide, and methane cycles. Many of these enzymes rely on Ni(I) oxidation states in critical catalytic intermediates. However, due to the highly reactive nature of these species, their isolation within metalloenzymes has often proved elusive. In this report, we describe and characterize a model biological Ni(I) species that has been generated within the electron transfer protein, azurin. Replacement of the native copper cofactor with nickel is shown to preserve the redox activity of the protein. The Ni(II/I) couple is observed at -590 mV versus NHE, with an interfacial electron transfer rate of 70 s(-1). Chemical reduction of Ni(II)Az generates a stable species with strong absorption features at 350 nm and a highly anisotropic, axial EPR signal with principal g-values of 2.56 and 2.10. Density functional theory calculations provide insight into the electronic and geometric structure of the Ni(I) species, suggesting a trigonal planar coordination environment. The predicted spectroscopic features of this low-coordinate nickel site are in good agreement with the experimental data. Molecular orbital analysis suggests potential for both metal-centered and ligand-centered reactivity, highlighting the covalency of the metal-thiolate bond. Characterization of a stable Ni(I) species within a model protein has implications for understanding the mechanisms of complex enzymes, including acetyl coenzyme A synthase, and developing scaffolds for unique reactivity.

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel­(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic NiIII-CH3 species initially generated can be rapidly reduced to a high-spin NiII-CH3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. NiIII-CH3 Az is only the second example of two-electron addition of methyl iodide to a NiI center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.

The biological global carbon cycle is largely regulated through microbial nickel enzymes, including carbon monoxide dehydrogenase (CODH), acetyl coenzyme A synthase (ACS), and methyl coenzyme M reductase (MCR). These systems are suggested to utilize organometallic intermediates during catalysis, though characterization of these species has remained challenging. We have established a mutant of nickel-substituted azurin as a scaffold upon which to develop protein-based models of enzymatic intermediates, including the organometallic states of ACS. In this work, we report the comprehensive investigation of the S = 1/2 Ni–CO and Ni–CH3 states using pulsed EPR spectroscopy and computational techniques. While the Ni–CO state shows conventional metal–ligand interactions and a classical ligand field, the Ni–CH3 hyperfine interactions between the methyl protons and the nickel indicate a closer distance than would be expected for an anionic methyl ligand. Structural analysis instead suggests a near-planar methyl ligand that can be best described as cationic. Consistent with this conclusion, the frontier molecular orbitals of the Ni–CH3 species indicate a ligand-centered LUMO, with a d9 population on the metal center, rather than the d7 population expected for a typical metal–alkyl species generated by oxidative addition. Collectively, these data support the presence of an inverted ligand field configuration for the Ni–CH3 Az species, in which the lowest unoccupied orbital is centered on the ligands rather than the more electropositive metal. These analyses provide the first evidence for an inverted ligand field within a biological system. The functional relevance of the electronic structures of both the Ni–CO and Ni–CH3 species are discussed in the context of native ACS, and an inverted ligand field is proposed as a mechanism by which to gate reactivity both within ACS and in other thiolate-containing metalloenzymes.

Edited by Ruma Banerjee Methanobactins (Mbns) are ribosomally-produced, post-translationally modified peptidic copper-binding natural products produced under conditions of copper limitation. Genes encoding Mbn biosynthetic and transport proteins have been identified in a wide variety of bacteria, indicating a broader role for Mbns in bacterial metal homeostasis. Many of the genes in the Mbn operons have been assigned functions, but two genes usually present, mbnP and mbnH, encode uncharacterized proteins predicted to reside in the periplasm. MbnH belongs to the bacterial diheme cytochrome c peroxidase (bCcP)/MauG protein family, and MbnP contains no domains of known function. Here, we performed a detailed bioinformatic analysis of both proteins and have biochemically characterized MbnH from Methylosinus (Ms.) trichosporium OB3b. We note that the mbnH and mbnP genes typically co-occur and are located proximal to genes associated with microbial copper homeostasis. Our bioinformatics analysis also revealed that the bCcP/MauG family is significantly more diverse than originally appreciated, and that MbnH is most closely related to the MauG subfamily. A 2.6 Å resolution structure of Ms. trichosporium OB3b MbnH combined with spectroscopic data and peroxidase activity assays provided evidence that MbnH indeed more closely resembles MauG than bCcPs, although its redox properties are significantly different from those of MauG. The overall similarity of MbnH to MauG suggests that MbnH could post-translationally modify a macromolecule, such as internalized CuMbn or its uncharacterized partner protein, MbnP. Our results indicate that MbnH is a MauG-like diheme protein that is likely involved in microbial copper homeostasis and represents a new family within the bCcP/MauG superfamily. Natural products that sequester and import vital and toxic metal ions play important roles in maintaining metal homeostasis in many species. Most well-studied are bacterial small molecules that bind ferric iron and are known as siderophores (iron "carriers") (1). In recent years, similar molecules that bind other metals have been discovered and characterized (2). One of these is a family of copper-binding compounds called methanobactins (Mbn) 5 (3, 4), which are produced from ribosomally synthesized peptides. All Mbns contain post-translational modifications that include nitrogen-containing heterocycles (oxazolones and pyrazinedione/diols) and neighboring thioamide/enethiol groups, and many have other less widespread modifications including "N-terminal" carbonyl groups (5, 6), intramolecular disulfide bonds (3, 6), and sulfonated threonines (7, 8). Of these functional groups, the N-heterocycles and thioamides provide ligands that chelate a copper ion. Mbns bind both Cu I and Cu II with high affinity (binding constants of 10 19-21 M Ϫ1 for Cu I and 10 11-14 M Ϫ1 for Cu II) (7-9); upon binding, the latter is quickly reduced to Cu I via an unknown mechanism. Under copper-limited conditions, some methanotrophs, bacteria that metabolize methane under...