The NAD linked soluble hydrogenase from Alcaligenes eutrophus H16: Detection and characterization of EPR signals deriving from nickel and flavin

In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme was either reduced with the natural electron carrier NADH (5-10 mM) or carefully titrated with sodium dithionite to an intermediate, narrow redox potential range (-280 to -350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at 80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments into three distinct signals: (1) the ''classical'' Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal (Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with Ni-61 (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved Ni-61 hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials determined at pH 7.0 for the described redox components were: for FMN -170 mV (midpoint potential, E(m) for appearance), -200 mV (EPR signal intensity maximum) and -230 mV (E(m) for disappearance); for the Ni centre (Ni-C), -290 mV (E(m) for appearance), -305 mV (signal in tensity maximum) and -325 mV (E(m) for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01.