Well-defined molecular systems for catalytic hydrogen production that are robust, easily generated, and active under mild aqueous conditions remain underdeveloped. Nickel-substituted rubredoxin (NiRd) is one such system, featuring a tetrathiolate coordination environment around the nickel center that is identical to the native [NiFe] hydrogenases and demonstrating hydrogenase-like proton reduction activity. However, until now, the catalytic mechanism has remained elusive. In this work, we have combined quantitative protein film electrochemistry with optical and vibrational spectroscopy, density functional theory calculations, and molecular dynamics simulations to interrogate the mechanism of H evolution by NiRd. Proton-coupled electron transfer is found to be essential for catalysis. The coordinating thiolate ligands serve as the sites of protonation, a role that remains debated in the native [NiFe] hydrogenases, with reduction occurring at the nickel center following protonation. The rate-determining step is suggested to be intramolecular proton transfer via thiol inversion to generate a Ni-hydride species. NiRd catalysis is found to be completely insensitive to the presence of oxygen, another advantage over the native [NiFe] hydrogenase enzymes, with potential implications for membrane-less fuel cells and aerobic hydrogen evolution. Targeted mutations around the metal center are seen to increase the activity and perturb the rate-determining process, highlighting the importance of the outer coordination sphere. Collectively, these results indicate that NiRd evolves H through a mechanism similar to that of the [NiFe] hydrogenases, suggesting a role for thiolate protonation in the native enzyme and guiding rational optimization of the NiRd system.

A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20-100 s(-1) at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.

Significance Methanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Nickel-substituted rubredoxin (NiRd) is a functional enzyme mimic of hydrogenase, highly active for electrocatalytic and solution-phase hydrogen generation. Spectroscopic methods can provide valuable insight into the catalytic mechanism, provided the appropriate technique is used. In this study, we have employed multi-wavelength resonance Raman spectroscopy coupled with DFT calculations on an extended active-site model of NiRd to probe the electronic and geometric structures of the resting state of this system. Excellent agreement between experiment and theory is observed, allowing normal mode assignments to be made on the basis of frequency and intensity analyses. Both metal-ligand and ligand-centered vibrational modes are enhanced in the resonance Raman spectra. The latter provide information about the hydrogen bonding network and structural distortions due to perturbations in the secondary coordination sphere. To reproduce the resonance enhancement patterns seen for high-frequency vibrational modes, the secondary coordination sphere must be included in the computational model. The structure and reduction potential of the NiIIIRd state have also been investigated both experimentally and computationally. This work begins to establish a foundation for computational resonance Raman spectroscopy to serve in a predictive fashion for investigating catalytic intermediates of NiRd.

Secondary sphere interactions are known to significantly impact catalytic rates within biological systems as well as synthetic molecular catalysts. The [NiFe] hydrogenase enzymes oxidize and produce molecular hydrogen at high turnover rates within a complex coordination environment. Nickel-substituted rubredoxin (NiRd) has been developed as a functional, protein-based mimic of the [NiFe] hydrogenase, providing an opportunity to understand the influence of the secondary coordination environment on proton reduction activity. In this work, a rationally designed series of mutants was generated to study the effects of outer-sphere interactions on catalysis. This library was characterized using quantitative protein film electrochemistry, optical spectroscopy, X-ray crystallography, and molecular dynamics simulations. Changing the secondary sphere residues modulates the redox activity of the nickel- and iron-bound rubredoxin proteins, alters the hydrogen-bonding network, and perturbs solvent accessibility of the active site, which correlates with catalytic turnover frequency. The effects on reactivity are dependent on the site of mutation and, when coupled to crystallographic and computational analyses, implicate one of the nickel-coordinating cysteine residues as the mechanistically relevant site of protonation. Introduction of a carboxylate residue, mimicking that found in the [NiFe] hydrogenase, significantly increases the overall catalytic rate, likely through installation of a proton transfer pathway into the active site. Apparent turnover frequencies within the mutant constructs range from 15 to 500 s–1 without imparting significant variation in overpotential, and many mutants break the typical scaling relationship between catalytic rates and overpotential that is often seen in small-molecule systems. These results demonstrate the substantial impact of the coordination environment on the hydrogen-producing activity of the artificial metalloenzyme, NiRd, and highlight the importance of such interactions within molecular catalysts.

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The use of biological systems for electrochemical energy conversion applications is often limited by instability of the protein or protein–electrode system. Here, we present a simple but efficient method for covalent attachment of nickel-substituted rubredoxin (NiRd), a model hydrogenase, to an unmodified graphite electrode based on amide bond formation. The resultant electrodes are shown to be highly active for H2 evolution over a period of several weeks. The effects of different attachment methods on interfacial electron transfer (ET) rates and catalysis are investigated, with decreased ET rates and increased background reactivity observed for surface-modified electrodes. Electrochemical simulations reveal that reduced protein dynamics of the attached NiRd enzyme are likely responsible for decreased catalytic rates by modulating the intramolecular proton transfer step. Ultimately, this straightforward approach can be broadly applied to diverse redox-active proteins and enzymes and will expand the utility of such systems by conferring increased stability over extended periods of time.

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates Nδ and Nω’ of Nω-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzyme’s C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbing µ-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among ∼9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, ∼25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases.Significance statementThe enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its central N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.