METHYL VIOLOGEN HYDROGENASE-II, A NEW MEMBER OF THE HYDROGENASE FAMILY FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM DELTA-H

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium th ermoautotrophicum DELTAH have been separated (resolution, R(s) at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M N aCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previo usly described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The sp ecific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of prot ein, respectively, when enzyme activity was compared at pH 7.5, the op timal pH for MVH II. Gel electrophoresis in nondenaturing systems indi cated that MVH I and MVH II had a similar molecular mass of 145 kDa. D enatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa ; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amin o acid sequences of the alpha, gamma, and delta subunits of MVH II wer e identical with the sequences of the equivalent subunits of MVH I. Ho wever, the N-terminal amino acid sequence of the beta subunit of MVH I I was totally different from the sequence of the beta subunit of MVH I . Both MVH I and MVH II had the same optimal temperature of 60-degrees -C for maximum activity. The pH optima of MVH I and MVH II were 9.0 an d 7.5, respectively. Most of the divalent metal ions tested significan tly inhibited MVH I activity, but MVH II activity was only partially i nhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at -20-degrees-C under anaerobic conditions. Wh en exposed to air, 90% of MVH I activity was lost within 2 min; howeve r, MVH II lost only 50% of its activity in 3 h.