The hydrogen binding site in hydrogenase: 35-GHz ENDOR and XAS studies of the nickel-C (reduced and active form) and the Ni-L photoproduct

Abstract

EPR, 35-GHz ENDOR, and X-ray absorption (XAS) spectroscopic studies of H2 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-ENDOR spectra reveal two sets of protons that interact strongly with the EPR-active center in Ni-C. The first proton set (HI) is solvent exchangeable, originates from dihydrogen, and has a value of A ( H 1 ) -20 MHz. The second proton set (H2) is not solvent exchangeable, has a coupling constant of A(H2) -12 MHz, and is assigned to one or more cysteine P-CH2 protons. Upon irradiation and conversion of the EPR signal to Ni-L, the exchangeable HI proton set is no longer observed. Upon annealing and regeneration of Ni-C, the feature associated with the H I 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.

Nickel hydrogenases: in search of the active site

“…Eidsness et al [56,57] concluded for the D. gigas enzyme that the coordination would be tetragonally-distorted octahedral or trigonalbipyramidal, in line with earlier conclusions based on the EPR spectra of Ni(III) in several hydrogenases [7,29,144]. Maroney and coworkers came to a similar conclusion from X-ray absorption studies on the T. roseopersicina enzyme [18,136,137,224,225].…”

“…Maroney and coworkers [225] have repeated and extended the ENDOR measurements with the T. roseopersicina enzyme displayed an isotropic coupling and were ascribed to /3-CH 2 protons from a cysteine residue [225].…”

“…Maroney and coworkers [18,136,137,224,225] have carried out extensive X-ray absorption measurements on the enzyme from T. roseopersicina. Their most recent interpretation for the Ni-C species is that 2 + 1 S(C1) at 2.22-2.25 ,~, 2 + 1 N(O) at 1.92-2.02 .~ and 1 distal S(CI) at 2.70-2.75 A are present [225] as ligands to nickel.…”

“…Their most recent interpretation for the Ni-C species is that 2 + 1 S(C1) at 2.22-2.25 ,~, 2 + 1 N(O) at 1.92-2.02 .~ and 1 distal S(CI) at 2.70-2.75 A are present [225] as ligands to nickel. In another publication of the same group [18], where five different states of the enzyme (from completely oxidized to completely reduced) were investigated, it was stated that EXAFS spectra obtained At that time, however, it was concluded that there was no distal S atom present in the Ni-C form.…”

“…In view of the widely different properties of nickel in the inactive and active state of the enzyme, it cannot be excluded from EPR measurements, though, that N is an equatorial ligand of Ni(III), where the unpaired spin, being in a d~2 orbital, probes only two directions. X-ray absorption measurements [18,57,136,137,224,225], however, argue against such a possible change.…”