Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

Abstract

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH.

Spectroscopic elucidation of energy transfer in hybrid inorganic–biological organisms for solar-to-chemical production

“…Inhibition of the H 2 ase active site (H cluster) with CO did not significantly change the TA kinetics, similar to previous works, in which e − transfer initially proceeds through the FeS cluster chain rather than directly to the NiFe active site (Fig. S2B) (20,31,32).…”

Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site

“…This is particularly clear in catalytic protein film voltammetry experiments, in which hydrogenase is adsorbed onto the surface of an electrode and catalytic current is measured as a function of potential, fingerprinting both the catalytic bias (ratio of H 2 oxidation to H 2 production current) and the potential at which catalysis commences ( Figure 1 b). 2 , 3 The [NiFe]-hydrogenases that are ideal bidirectional H 2 electrocatalysts, displaying high H 2 production and oxidation turnover rates, are inactivated by O 2 (O 2 -sensitive, e.g., Escherichia coli hydrogenase-2). 2 , 4 , 5 Conversely, the O 2 -tolerant MBHs are poor H 2 -producing catalysts and require an additional thermodynamic driving force (overpotential) to initiate H 2 oxidation at pH > 5, e.g., Escherichia coli hydrogenase-1 ( E. coli Hyd-1).…”

Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane‐Bound [NiFe] Hydrogenase‐1

“…These H 2 ‐enzymes are particularly amenable to study by protein film electrochemistry (PFE) because the rapid rate of electrocatalytic turnover acts as a highly effective signal amplifier, meaning that substantial levels of catalytic current can be detected even when only a small number of molecules have adsorbed to the electrode surface in an electroactive configuration (typical electroactive coverages are in the region of 7 pmol cm −2 ) . When coupled with structural and molecular biology mutation studies, this electrochemical technique becomes a powerful assay method that can help pinpoint the role of certain amino acids in tuning the chemistry of a hydrogenase . In particular, the energetic efficiency of catalysis, often quantified as the “overpotential” voltage difference between the onset of catalysis and the experimental hydrogen reduction potential, E (H + /H 2 ), and the catalytic “bias” (ratio of H 2 ‐oxidation to H + ‐reduction current) are readily quantified in PFE.…”

“…In O 2 ‐sensitive [NiFe] hydrogenases such as E. coli Hyd‐2, the proximal cluster is a standard ferredoxin‐like [Fe 4 S 4 ] 2+/1+ center . However, crystal structures of O 2 ‐sensitive [NiFe] hydrogenases also show a large‐subunit His residue close to the proximal cluster,, and amino acid sequence comparisons identify this highly conserved residue as HydC‐His214 in E. coli Hyd‐2 (Figure ). Based on the Dance mechanism, it is postulated that removal of this His residue should not have a dramatic impact on enzymatic reactivity, because Hyd‐2 does not contain a [Fe 4 S 3 Cys 2 ] proximal cluster.…”

“…At pH 6.0 both native Hyd‐2 and the Hyd2‐H214A variant exhibit the characteristic aerobic reactivity of O 2 ‐sensitive hydrogenases; upon addition of 3 % O 2 into the gas mixture their catalytic activity tends towards zero (the native enzyme reaches 0 mA while the variant is at 4 % of the initial oxidation activity after 5 min exposure to a 3 % O 2 , 3 % H 2 gas mixture). When O 2 is removed from the gas stream, there is no significant difference in the extent of reactivation of native Hyd‐2 and Hyd2‐H214A, suggesting that the His does not act in a protective manner against formation of the slow‐reactivating, O 2 ‐inihibited Ni−A state which O 2 ‐sensitive [NiFe] hydrogenases are known to form ,,…”