Hydrogen Isotope Effects in the Reactions Catalyzed by H₂‐forming N⁵,N¹⁰‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea

Abstract

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

Carbon and hydrogen isotope fractionation by moderately thermophilic methanogens 1 1Associate editor: N. E. Ostrom

“…Hydrogen from H 2 is affixed directly to the growing methane moiety, though isotope exchange occurs (Schworer et al, 1993;Klein et al, 1995a,b;Hartmann et al, 1996). There is no deuterium isotope effect associated with this reaction (Klein et al, 1995b).…”

“…This enzyme catalyzes the heterolytic cleavage of H 2 and the subsequent hydride shift to a conjugated carbocation reaction center (Schleucher et al, 1994;Klein et al, 1995b;Hartmann et al, 1996;. Hydrogen from H 2 is affixed directly to the growing methane moiety, though isotope exchange occurs (Schworer et al, 1993;Klein et al, 1995a,b;Hartmann et al, 1996).…”

“…The mass balance is detailed in section 2.4.4. Although one uptake hydrogenase (H 2 -forming N 5 ,N 10 -methylenetetrahydromethanopterin dehydrogenase, as in section 3.2.2.2) displays no fractionation during H 2 uptake (e.g., ␣ ϭ 1; Klein et al, 1995b), it is likely other uptake hydrogenases in M. marburgensis exhibit greater fractionation factors. Figure 13a displays the relationship between the isotopic fractionation for H 2 production, ␣(H 2 O-H 2 ), and the efficiency of the hydrogenase system (E), at three different values of ␣ c .…”

Methanogenesis Biochemistry

“…The enzyme catalyses the stereospecific transfer of a hydride ion from H 2 into the pro-R position of the methylene carbon of CH H 4 MPT 1 . A stereospecific exchange of the pro-R hydrogen with water led to the proposal that CH H 4 MPT 1 reduction with H 2 involves a pentacoordinated carbonium ion (CH 3 1 =H 4 MPT) transition state intermediate where the pro-R hydrogen bond is protonated and exchanges with water (Klein et al, 1995).…”

Structure and Function of [Fe]‐Hydrogenase and its Iron–Guanylylpyridinol (FeGP) Cofactor

“…[12,13] The open site could also be used as an alternative binding site for the second CO ligand. [7] [Fe]-hydrogenase catalyzes a single and double exchange between D 2 [14][15][16] and protons of bulk water and a conversion of para-H 2 into ortho-H 2 , both only in the presence of methenyl-H 4 MPT + . [17] Thus, H 2 activation necessarily requires the substrate.…”