INTERACTIONS OF SE-77 AND (CO)-C-13 WITH NICKEL IN THE ACTIVE-SITE OFACTIVE F-420-NONREDUCING HYDROGENASE FROM METHANOCOCCUS-VOLTAE

The selenium-containing F-420-nonreducing hydrogenase from Methanococc us voltae was prepared in the Ni-a(I). CO state. The effect of illumin ation on this light-sensitive species was studied. EPR studies were ca rried out with enzyme containing natural selenium or with enzyme enric hed in Se-77. Samples were prepared with either CO or (CO)-C-13. In th e Ni-a(I). CO state, the nuclear spins of both Se-77 (I = 1/2) and C-1 3 (I = 1/2) interacted with the nickel based unpaired electron, sugges ting that they are positioned on opposite sites of the nickel ion, In the light-induced signal, the interaction with (CO)-C-13 was lost. The Se-77 nuclear spin introduced an anisotropic hyperfine splitting in b oth the dark and light-induced EPR signals, The data on the active enz yme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Char on, M. H., Piras, C, Hatchikian, E. C., Frey, M., and Fontecilla Camps , J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.