ISSN:
1432-1327
Keywords:
Key words Alcaligenes eutrophus
;
Hydrogenase
;
EPR spectroscopy
;
Nickel centre
;
Iron-sulfur cluster
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract 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 61Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved 61Ni 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, Em, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (Em for disappearance); for the Ni centre (Ni-C), –290 mV (Em for appearance), –305 mV (signal intensity maximum) and –325 mV (Em 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s007750050028
