Insights into electron transfer and bifurcation of the Synechocystis sp. PCC6803 hydrogenase reductase module

Abstract

The NAD⁺-reducing soluble [NiFe] hydrogenase (SH) is the key enzyme for production and consumption of molecular hydrogen (H₂) in Synechocystis sp. PCC6803. In this study, we focused on the reductase module of the SynSH and investigated the structural and functional aspects of its subunits, particularly the so far elusive role of HoxE. We demonstrated the importance of HoxE for enzyme functionality, suggesting a regulatory role in maintaining enzyme activity and electron supply. Spectroscopic analysis confirmed that HoxE and HoxF each contain one [2Fe2S] cluster with an almost identical electronic structure. Structure predictions, alongside experimental evidence for ferredoxin interactions, revealed a remarkable similarity between SynSH and bifurcating hydrogenases, suggesting a related functional mechanism. Our study unveiled the subunit arrangement and cofactor composition essential for biological electron transfer. These findings enhance our understanding of NAD⁺-reducing [NiFe] hydrogenases in terms of their physiological function and structural requirements for biotechnologically relevant modifications.