STABILITY OF THE NI-C STATE AND OXIDATIVE TITRATIONS OF DESULFOVIBRIO-GIGAS HYDROGENASE MONITORED BY EPR AND ELECTRONIC ABSORPTION SPECTROSCOPIES

The Ni-C state of the NiFe hydrogenase from Desulfovibrio gigas was fo und to be stable for over 40 h at pH 8.0 in the strict absence of H-2. This demonstrates that Ni-C is unable to spontaneously reduce protons to H-2 under these conditions, a result that is contrary to earlier r eports. The form of the nickel that spontaneously reduces protons is p robably Ni-R, the EPR-silent state that appears to be one electron mor e reduced than Ni-C, The stability of Ni-C permitted, for the first ti me, stoichiometric oxidative titrations of the H-2-free, reduced enzym e. Four such titrations were performed, using the oxidant thionin and monitoring the progress of the titrations by electronic absorption (at 410 nm) and EPR spectroscopies. Redox changes in the enzyme's two Fe4 S4 clusters were readily followed by changes at 410 nm, while those of the Ni were observed by EPR. Redox changes of the Fe3S4 cluster were monitored by both spectroscopic methods. At the start of the titration s, the Ni center was in the Ni-C state and the Fe-S clusters were part ially oxidized. Adding thionin caused disappearance of Ni-C, developme nt of the EPR-silent intermediate state, and eventually the appearance of Ni-B. The Fe-S clusters oxidized gradually throughout every stage of the titrations. An average of 4.2 oxidizing equiv/mol of thionin wa s consumed overall. After the number of equiv/mol consumed by the Fe-S clusters and the EPR-active Ni ions were substracted, an average of 1 .2 oxidizing equiv/mol remained unassigned. The activity of the enzyme appears to be correlated to the ability of the Ni site to undergo red ox chemistry.