THE HYDROGEN BINDING-SITE IN HYDROGENASE - 35-GHZ ENDOR AND XAS STUDIES OF THE NI-C ACTIVE FORM AND THE NI-L PHOTOPRODUCT

EPR, 35-GHz ENDOR, and X-ray absorption (XAS) spectroscopic studies of H-2 binding in hydrogenase from Thiocapsa roseopersicina are reported. These studies involve spectra taken on an active form of the enzyme that displays an EPR signal designated Ni-C. This form of the enzyme is light sensitive and is converted to a photoproduct that exhibits a unique EPR signal, Ni-L. H-1-ENDOR spectra reveal two sets of protons that interact strongly with the EPR-active center in Ni-C. The first proton set (H1) is solvent exchangeable, originates from dihydrogen, and has a value of A(H1) is similar to 20 MHz. The second proton set (H2) is not solvent exchangeable, has a coupling constant of A(H2) is similar to 12 MHz, and is assigned to one or more cysteine beta-CH2 protons. Upon irradiation and conversion of the EPR signal to Ni-L, the exchangeable H1 proton set is no longer observed. Upon annealing and regeneration of Ni-C, the feature associated with the H1 proton set is again observed. This is direct evidence that the photoprocess and annealing involve dissociation of a hydrogenic proton and recombination. XAS spectra taken on samples poised to maximize Ni-C and Ni-L do not reveal any significant structural difference between the Ni sites.