Functional roles of the [2Fe-2S] clusters in Synechocystis PCC 6803 Hox [NiFe]-hydrogenase reactivity with ferredoxins.

The HoxEFUYH complex of Synechocystis PCC 6803 (S. 6803) consists of a HoxEFU ferredoxin:NAD(P)H oxidoreductase subcomplex and a HoxYH [NiFe]-hydrogenase subcomplex that catalyzes reversible H₂ oxidation. Prior studies have suggested that the presence of HoxE is required for reactivity with ferredoxin; however, it is unknown how HoxE is functionally integrated into the electron transfer network of the HoxEFU:ferredoxin complex. Deciphering electron transfer pathways is challenged by the rich iron-sulfur cluster content of HoxEFU, which includes a [2Fe-2S] cluster in each subunit, along with multiple [4Fe-4S] clusters and a flavin cofactor. To resolve the role of HoxE, we determined the biophysical and thermodynamic properties of each [2Fe-2S] cluster in HoxEFU using steady-state and potentiometric EPR analysis in combination with square wave voltammetry (SWV). The temperature-dependence of the EPR signal for HoxE confirmed the coordination of a single [2Fe-2S] cluster that was shown by SWV to have an E_m = -424 mV (versus SHE). Strikingly, when the E_m of the HoxE [2Fe-2S] cluster was analyzed in HoxEFU titrations, it was shifted by >100 mV to an E_m < -525 mV (versus SHE). EPR titrations of HoxEFU gave an E_m value for the [2Fe-2S] cluster of HoxF, E_m = -419 mV and HoxU, E_m = -349 mV. These values were used to re-analyze the diaphorase kinetics in reactions performed with ferredoxins with varying E_m's. The results are formulated into a model of HoxEFU:ferredoxin reactivity and the role of HoxE in mediating electron transfer within the HoxEFU:ferredoxin complex.