Background: Hydrogenases contain a unique oxygen-labile metal cofactor. Results: Substitution of noncovalently interacting residues degrades the catalytic cofactor (K358N and M497L) or reduces activity but leaves the cofactor chemically intact (C299S and M353L

This discussion describes efforts to produce a stable, efficient electrocatalyst for four-electron O2 reduction through the direct attachment of fungal laccase, a 'blue' copper oxidase, to functionalised carbon electrode materials. Commercially available carbons, including fibrous and porous materials, offer important opportunities for achieving high conductivity over high surface areas that can be chemically functionalised. A promising approach for attaching laccase to a carbon surface is to use the diazonium coupling reaction to generate protrusive aromatic functionalities that can bind to hydrophobic residues close to the 'blue' Cu site: this site provides a fast, intramolecular electron relay into the buried trinuclear Cu active site that converts O2 rapidly and cleanly to H2O. This enhancement procedure makes possible the stable, direct electrocatalytic reduction of O2 at high potential with high efficiency in terms of turnover frequency per enzyme active site engaged with the electrode. The absence of electron-transfer mediators and simplicity of electrode system reveals the more inherent characteristics of the electrocatalytic mechanism that are masked in the waveform when a mediator is used. The study includes experiments to assess the effects of methanol and chloride ions on laccase electrocatalysis, complementing studies carried out by other groups, particularly those in which laccase is embedded in an electron-mediating gel.

Formaldehyde-a rapid and reversible inhibitor of hydrogen evolution by [FeFe]-hydrogenases-binds with a strong potential dependence that is almost complementary to that of CO. Whereas exogenous CO binds tightly to the oxidized state known as H(ox) but very weakly to a state two electrons more reduced, formaldehyde interacts most strongly with the latter. Formaldehyde thus intercepts increasingly reduced states of the catalytic cycle, and density functional theory calculations support the proposal that it reacts with the H-cluster directly, most likely targeting an otherwise elusive and highly reactive Fe-hydrido (Fe-H) intermediate.