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.

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.

An enzymatic system for light-driven hydrogen generation has been developed throughc ovalent attachment of ar uthenium chromophore to nickel-substituted rubredoxin (NiRd).The photoinduceda ctivity of the hybrid enzymei ss ignificantly greater than that of at wo-component system and is strongly dependent on the positiono ft he ruthenium phototriggerr elative to the active site, indicating ar ole for intramolecular electron transfer in catalysis. Steady-state and time-resolved emission spectra reveal ap athway for rapid, directq uenching of the ruthenium excited state by nickel, butl ow overall turnover numberssuggest initial electron transfer is not the rate-limiting step. This approach is ideally suited for detailed mechanistic investigationso fc atalysis by NiRd and other molecular systems, with implications for generation of solar fuels.Hydrogeni sc onsidered to be ac lean, renewable alternative to carbon-based fuels. However, generating hydrogen through traditional methods such as electrolytic water splitting can be costly and energy intensive, thus overriding the advantages of this supposedly sustainable fuel. [1][2][3] In contrast to anthropogenic approaches, biological systems have evolvedt op roduce hydrogen under mild conditions by using proteins called hydrogenases. [4,5] Although theseh ighly efficient enzymes suffer from extreme fragilityu nder atmospheric conditions, precluding general application, they have inspired the development of chemically diverse hydrogenase models. [6][7][8][9][10][11][12][13][14][15][16][17][18] Of particulari nterest is the design of systemst hat can be driven by light for the generation of so-called "solar fuels". [19][20][21][22][23][24][25] We have shown that nickel-substituted rubredoxin (NiRd),asmall electron-transfer protein that naturally binds iron in at etrahedral tetrathiolate coordination motif, mimics the structure and function of the redox-active site in the [NiFe] hydrogenases. The hydrogenevolvinga ctivity of this system has previously been characterized by using solution-based photochemical and electrocatalytic assays. [26] In this study,aruthenium chromophore was covalently attachedt oN iRd to directlyr educe the active site for light-initiated catalysis. Attachment at different locations aroundt he protein surface reveals as trong distance dependence for hydrogen evolution activity,a nd time-resolved emission studies were used to investigate the efficiency of electron transfer between the ruthenium and nickel centers. Low overall turnover numbers across all of the NiRd variants suggest initial electron transfer is not the rate-limiting step in catalysis.The ruthenium chromophore [Ru II (2,2'-bipyridine) 2 (5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline)] 2 + was synthesized as previously reported and bound to the sulfhydryl group of af ree cysteiner esidue. [27] Following purification,t he labeling efficiency was found to be 90 AE 5% by optical absorption (see the Supporting Information, Figure S1). MALDI-TOF mass spectrometry confirmed this yield, showing ...

The life-sustaining reduction of N2 to NH3 is thermoneutral yet kinetically challenged by high-energy intermediates such as N2H2. Exploring intramolecular H-bonding as a potential strategy to stabilize diazene intermediates, we employ a series of [ xHetTpCu]2(μ-N2H2) complexes that exhibit H-bonding between pendant aromatic N-heterocycles (xHet) such as pyridine and a bridging trans-N2H2 ligand at copper­(I) centers. X-ray crystallography and IR spectroscopy clearly reveal H-bonding in [pyMeTpCu]2(μ-N2H2) while low-temperature 1H NMR studies coupled with DFT analysis reveals a dynamic equilibrium between two closely related, symmetric H-bonded structural motifs. Importantly, the xHet pendant negligibly influences the electronic structure of xHetTpCuI centers in xHetTpCu­(CNAr2,6‑Me2 ) complexes that lack H-bonding as judged by nearly indistinguishable ν­(CN) frequencies (2113–2117 cm–1). Nonetheless, H-bonding in the corresponding [ xHetTpCu]2(μ-N2H2) complexes results in marked changes in ν­(NN) (1398–1419 cm–1) revealed through resonance Raman studies. Due to the closely matched N–H BDEs of N2H2 and the pyH0 cation radical, the aromatic N-heterocyclic pendants may encourage partial H-atom transfer (HAT) from N2H2 to xHet through redox-non-innocent H-bonding in [ xHetTpCu]2(μ-N2H2). DFT studies reveal modest thermodynamic barriers for concerted transfer of both H-atoms of coordinated N2H2 to the xHet pendants to generate tautomeric [ xHetHTpCu]2(μ-N2) complexes, identifying metal-assisted concerted dual HAT as a thermodynamically favorable pathway for N2/N2H2 interconversion.