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.

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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