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 (vs 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 (vs 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.