Power of the Secondary Sphere: Modulating Hydrogenase Activity in Nickel-Substituted Rubredoxin

Abstract

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.

Biosynthetic Approaches towards the Design of Artificial Hydrogen‐Evolution Catalysts

“…Based on studies from a series of secondary sphere NiRd mutants it was found that the mutations modulated the activity, H‐bonding network and active site solvent accessibility. The mutant library was made by mutagenesis of three residues in the metal binding loops, Val8, Val34 and Val37.…”

Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors

“…50 Photocatalytic activity could be achieved and modulated by selective conjugation of a Ru(bpy)3 complex, 51 and detailed spectroscopic and computational characterization of the model elucidated a thiol-inversion mechanism of Ni(III)-hydride formation that is directly applicable to native [NiFe] hydrogenase. 52,53 In addition to mononuclear metalloproteins, heteronuclear bimetallic enzymes have also benefited from replacing the native with non-native metal ions. Despite highly homologous active sites, why a nonheme Cu atom was selected for O 2 reduction in HCO but nonheme Fe was selected for NO reduction in NOR had never been directly probed because removing the nonheme metal ion of either enzyme destabilizes it.…”

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].…”

“…A TON greater than 100 (nmol/mg) catalyst was observed, and the biomimetic catalyst is stable through catalysis in aqueous solution, allowing greater than 90% catalyst recovery. The protein structure is amenable to many different mutations, opening the possibility for optimization [160]. In further work, a ruthenium sensitizer was covalently attached to the protein scaffold, creating an integrated hydrogen evolution system [159].…”