Decoding the Elusive Redox Properties of [FeS] Clusters in [FeFe]-Hydrogenase on a Nanostructured Electrode

Proton-coupled electron transfer (PCET) is essential for [FeFe]-hydrogenase to operate reversibly with high activity at a minimal overpotential. However, whether the reduction of the [4Fe4S] cluster is proton-coupled during the catalytic cycle remains under debate. Here, we employ indium tin oxide (ITO)-functionalized nanostructured electrodes to resolve the elusive non-turnover redox signals of all three [4Fe4S] clusters in <i>Desulfovibrio desulfuricans</i> [FeFe]-hydrogenase (<i>Dd</i>HydAB). Analysis of the redox properties of the [4Fe4S] clusters revealed a moderate pH dependence (∼30 mV/pH), suggesting that the reduction potential is modulated by second-coordination-sphere effects rather than direct protonation of the coordinating cysteine residues. Additionally, integration of the non-turnover signals yielded an accurate quantification of the enzyme's concentration on the electrode surface. In combination with <i>in situ</i> maturation, this allowed us to quantitatively determine the turnover frequency of the enzyme on the electrode surface.