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 Desulfovibrio desulfuricans [FeFe]-hydrogenase (DdHydAB). 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 in situ maturation, this allowed us to quantitatively determine the turnover frequency of the enzyme on the electrode surface.