The Mechanism of [FeFe]-Hydrogenases—How Aldehydes Inhibit H₂ Evolution

Abstract

This chapter deals with investigations into the mechanism of [FeFe]-hydrogenases, using aldehyde molecules as (partly) reversible inhibitors. Of particular interest is the order of the electron and proton transfer steps that lead to the formation of hydrogen. Building upon the initial discovery of formaldehyde being a reversible inhibitor of [FeFe]-hydrogenases that preferably targets the super-reduced oxidation level of this class of enzymes [1, 2], the reaction of [FeFe]-hydrogenases with four different aldehydes of varying steric demand and electrophilicity is studied by protein film electrochemistry techniques. The degree of inhibition seems to correlate with the steric nature of the aldehydes and their reactivities towards nucleophiles. Analysis of the potential dependences and reversibility patterns of inhibition suggests that the smaller aldehydes react directly at the H-cluster, corroborating earlier findings made with formaldehyde [1, 2]. To gain structural insight into the binding of aldehydes to the H-cluster, inhibition by formaldehyde, the simplest and (due to its highly electrophilic carbonyl group) most reactive aldehyde inhibitor of the H-cluster, is studied by pulsed-EPR techniques in collaboration with Dr. William Myers (Centre for Advanced Electron Spin Resonance, University of Oxford). ESEEM experiments on deuterated formaldehyde firmly establish that formaldehyde can access the enzyme active site. ENDOR studies using carbon-13-labelled formaldehyde give rise to hyperfine interactions that are in agreement with several binding modes. Complementary density functional theory calculations by Professor John McGrady (Inorganic Chemistry Laboratory, University of Oxford) provide a mechanism that accounts for specific binding of formaldehyde to the distal iron of the H-cluster. A comparative analysis of the experimental evidence provided by different techniques leads to the proposal of a new mechanism for formaldehyde inhibition with important implications on the catalytic cycle: unusual for biological systems, protonation of the H-cluster only takes place after the consecutive transfer of two electrons.