Studies of light-induced nickel EPR signals in hydrogenase: comparison of enzymes with and without selenium

Abstract

Upon reduction under hydrogen-argon atmosphere, the nickel-hydrogenases generally show a characteristic rhombic EPR spectrum which is known as NiC. Illumination of this state at temperatures below 60 K has previously been shown to cause the disappearance of the Ni-C signal and the simultaneous appearance of two overlapping signals, here referred to as NiL1 and NiL2, which revert to the Ni-C state at higher temperatures. These phenomena have been compared in three nickel-containing hydrogenases, the [NiFe]-hydrogenases from Desulfovibrio gigas and Desulfovibrio fructosouorans, and the selenium-containing soluble [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. Significant differences were observed between these enzymes. (1) The NiC, NiL1 and NiL2 EPR spectra were almost identical for D. gigas and D. fructosouorans hydrogenases, but the rates of photoconversion of NiC to NiL1 were different, being about 5 times slower for D. fructosouorans than for D. gigas in H 2O. (2) The kinetic isotope effect in 2H 20/H 20 was a factor of thirty in D. gigas, but only two in D. fructosouorans. Dm. baculatum hydrogenase showed almost no kinetic isotope effect on the NiC to NiL1 conversion, but an effect on the conversion of NiL1 to NiL2. The kinetic isotope effects indicate that the processes involve the movement of a hydrogen nucleus. (3) The NiL1 species converted to into NiL2 in the dark at a rate that was virtually temperature-independent below 30 K, indicative of a proton tunnelling process. (4) The conversion of NiL1 to NiL2 was partly reversed by light in Dm. baculatum hydrogenase, but not in the [NiFe]-hydrogenases. (5) Prolonged illumination of the three enzymes induced the appearance of a third light-induced signal, NiL3. The new signal was rhombic, with features at g = 2.41, 2.16 (the third component being unresolved) in the [NiFe]-hydrogenases and g = 2.48, 2.16, 2.03 for the [NiFeSe]-enzyme. (6) Splittings caused by by spin-spin interactions with [4Fe-4S] clusters were detected for all the illuminated signals, NiL1, NiL2 and NiL3. These were quantitatively different for the three enzymes. (7) Broadening of the NiC signals in H 2O compared with 2H 20 was observed in the g 1 and g 2 components of D. gigas and D. fructosovorans hydrogenases, but not for Dm. baculatum. This broadening effect was not seen with any of the NiL species. These comparative effects are discussed in terms of subtle differences in the structure and protein environment of the nickel site, and access to exchangeable hydrons.