Cryo-EM map and functional electron transport studies of the HoxEFU subcomplex of the HOX [NiFe]-hydrogenase from Synechocystis sp. PCC 6803

The HOX [NiFe]-hydrogenase of Synechocystis sp. PCC 6803 is composed of large (HoxH) and small (HoxY) subunits that catalyze H 2 activation and HoxE, HoxF, and HoxU subunits that catalyze reactions with NAD(H), ferredoxins, or flavodoxin. Here, we report single-particle cryoelectron microscopy (cryo-EM) and electron paramagnetic resonance (EPR) spectroscopy for kinetic modeling of electron transfer in the HoxEFU subcomplex. Cryo-EM of resting-state HoxEFU yielded a low-resolution map with space for a dimer of HoxEFU monomers. An AlphaFold model of the dimer identified inter-cofactor distances and was used with cofactor E m values determined here to calculate electron transfer rate constants, k ET , for individual steps. We used these values to model electron transfer kinetics for HoxEFU NADH oxidation, which reproduced the equilibrium populations of reduced cofactors observed in EPR spectra. Overall, the results are interpreted in terms of how HoxEFU functions to coordinate electron transfer in reactions with NADH and ferredoxin.