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Abstract

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N10-methenyltetrahydomethanopterin (CH≡H4MPT4) with H2 to N5,N10-methylenetetrahydromethanopterin (CH2=H4MPT) and a proton (?G°?=? 5.5 kJ/mol). The enzyme also catalyzes a CH≡H4MPT+-dependent H2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH≡H4MPT+ reduction was performed with D2 instead of H2, Vmax and the Km did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H2O or D2O was the solvent. The findings indicate that a step other than the activation of H2 was rate-determining in CH≡H4MPT+ reduction with H2. This was substantiated by the observation that also the CH≡H4MPT+-dependent H2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. VMax for CH2=H4MPT dehydrogenation to CH≡H4MPT+ and H2 was only 2?3 times higher than for CD2=H4MPT dehydrogenation to CD≡H4MPT+ and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2=H4MPT was only rate-limiting when hydrogen was substituted by a deuterium.