Thermodynamic landscape of the redox-centres in the electron-confurcating [FeFe]-Hydrogenase (TmHydABC) of Thermotoga maritima

The heterotrimeric [FeFe]‑hydrogenase (TmHydABC) from Thermotoga maritima synergistically uses electrons from ferredoxin and NADH to reduce protons to hydrogen enabling the anaerobic metabolism of carbohydrates and other molecules. The precise mechanism by which this flavin-based electron confurcation is achieved is still unknown. Here, the redox properties of the cofactors in TmHydABC were characterized via chemical and electrochemical redox titrations monitored by EPR- and UV/Vis-spectroscopy. Deconvolution of nine out of eleven iron‑sulfur clusters harboured by the apo-enzyme was achieved by (a) exploiting the distinct relaxation properties (and hence temperature-dependencies) of the EPR-signals, (b) making use of spectral idiosyncrasies of [4Fe-4S] versus thioredoxin-like [2Fe-2S] clusters in UV/Vis-spectroscopy and (c) using the individual HydA, HydB and HydC subunits and C-terminal domains of HydA and HydB. Furthermore, electrostatic interactions between certain neighbouring clusters and a paramagnetic interaction between the flavin and one of the iron‑sulfur clusters were revealed. The electrochemical parameters of the flavin were obtained both via UV/Vis-spectroscopy and EPR-analysis of its semi-reduced state in isolated HydB. This semi-reduced state was found to correspond to a neutral flavosemiquinone and a pK_a (attributed to the N5-proton) was extracted from the pH-dependence of its redox midpoint potential. The electrochemical properties of the flavin in the entire enzyme appear to differ significantly from those in the isolated HydB subunit. The flavin and iron‑sulfur cluster redox properties indicate a dynamic electrochemical landscape, potentially contributing to its mechanism.