THE REDOX PROPERTIES AND ACTIVATION OF THE F420-NON-REACTIVE HYDROGENASE OF METHANOBACTERIUM-FORMICICUM

The F420-non-reactive hydrogenase of Methanobacterium formicicum (strain MF) is a dimeric protein of 70 kDa containing approx. 10 Fe, 8 S2-, 1 Ni, 1 Zn and 2 Cu atoms/mol. The air-oxidized hydrogenase purified under aerobic conditions did not catalyze H2 oxidation and catalyzed H2 evolution with a lag phase. The activated enzyme, characterized by maximal H2 oxidation activity and no lag phase in H2 production, was only obtained by incubating the enzyme at a redox potential below -400 mV (pH 8) in the presence of an electron carrier (methyl or benzyl viologen). The activated enzyme was irreversibly inactivated at redox potentials of greater than -100 mV. Chelator studies suggested that the activation process involved the incorporation of metal ions. Air-oxidized hydrogenase (as isolated) exhibited three distinct EPR signals which were assigned to a [3Fe-xS] cluster (g = 2.01, Em8 -75 mV), a Ni center (g = 2.29, 2.23 (2.22) and 2.01, Em8 -410 mV), and ''free'' Fe3+ (g = 4.30). The signals from the 3Fe- and Ni-centers were lost by reduction with H2 and were replaced by a broad g = 1.94 signal indicative of reduced [4Fe-4S] clusters. Upon activation of the enzyme, the g = 4.30 signal disappeared. None of the EPR signals observed in the oxidized enzyme reappeared upon controlled oxidation of the activated enzyme. Instead, an intense EPR signal centered at g = 2.06 was observed (Em8 0 mV) which was assigned to a Cu(II) center. These data suggest a possible role for copper in hydrogenase and that reductive activation involves (a) some 3Fe- to 4Fe-cluster conversion and (b) the irreversible reductive modification of a Ni-center.