Going beyond Structure: Nickel-Substituted Rubredoxin as a Mechanistic Model for the [NiFe] Hydrogenases

Abstract

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.

Biosynthetic Approaches towards the Design of Artificial Hydrogen‐Evolution Catalysts

“…The mechanism of H 2 evolution in NiRd was probed by protein film electrochemistry (PFE), spectroscopy, and computational studies . The non‐catalytic Fe III/II redox couple was used as an internal standard with NiRd to obtain the total electroactive protein coverage and quantitative information from the PFE studies.…”

Robust Carbon-Based Electrodes for Hydrogen Evolution through Site-Selective Covalent Attachment of an Artificial Metalloenzyme

“…Desulfovibrio desulfuricans ATCC 27774 wild-type Rd and all mutants were heterologously expressed, purified, and metalated, as previously described. − Protein purity was confirmed by native gel electrophoresis following metal substitution.…”

“…A 2 μL aliquot of the Fe II Rd protein sample was oxidized by the addition of 5 mM K 3 [Fe­(CN) 6 ] under ambient conditions for concentration determination using optical spectroscopy. An extinction coefficient of 7800 M –1 cm –1 at 492 nm for Fe III Rd was used …”

“…The protein fold is remarkably resistant to a wide range of pH values, temperatures, solvents, and oxygen, as well as straightforward to express and stable toward mutagenesis . Although the protein natively binds iron (FeRd), it has been previously demonstrated that the active-site metal can be easily replaced with a variety of different transition metals, such as nickel, zinc, copper, and cobalt, without compromising the stability or protein fold. − This facile metal exchange renders Rd a particularly attractive system for the engineering of an artificial hydrogenase; the protein coordination environment of nickel-substituted rubredoxin (NiRd) closely resembles the active site structure at the nickel center of the [NiFe] H 2 ase. ,, Prior work by our group and others has shown that NiRd serves not only as a structural mimic but also as a functional and mechanistic model enzyme for the [NiFe] H 2 ase. ,− Initial characterization of NiRd demonstrated that the protein is catalytically active toward hydrogen production . Detailed mechanistic studies on the NiRd metalloenzyme resolved intrinsic electrocatalytic properties of the active site, metrics that are masked by the iron–sulfur cluster electron relay in the native enzyme .…”

Light‐driven catalysis with engineered enzymes and biomimetic systems

“…Although most hydrogenase mimicry has focused on the diiron hydrogenase, one of the earliest systems was based on the [NiFe] hydrogenase [155][156][157][158][159][160][161]. Substitution of Ni in rubredoxin yields a catalyst, Ni-Rd, with a tetrathiolate active site mimicking the primary coordination sphere of the Ni site in [NiFe] hydrogenase [155,161].…”