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

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 fructosovorans, 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. fructosovorans hydrogenases, but the rates of photoconversion of Ni-C to Ni-L1 were different, being about 5 times slower for D. fructosovorans than for D. gigas in H2O. (2) The kinetic isotope effect in (H2O)-H-2/H2O was a factor of thirty in D. gigas, but only two in D. fructosovorans. 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 Ni-L2 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 H2O compared with (H2O)-H-2 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.