H sub(2)-forming N super(5),N super(10)-methylenetetra-hydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum . Studies of the catalytic mechanism of H sub(2) formation using hydrogen isotopes

• Published in: European journal of biochemistry Vol. 212; no. 1; pp. 255 - 261
• Main Authors: Schwoerer, B, Fernandez, V M, Zirngibl, C, Thauer, R K
• Format: Journal Article
• Language: English
• Published: 01.01.1993
• Subjects: Methanobacterium thermoautotrophicum
• ISSN: 0014-2956

H sub(2)-forming N super(5),N super(10)-methylenetetra-hydromethanopterin dehydrogenase is a novel hydrogenase found in most methanogenic archaea. It catalyzes the reversible conversion of N super(5),N super(10)-methylenetetra-hydromethanopterin (CH sub(2) = H sub(4)MPT) to N super(5),N super(10)-methenyltetra-hydromethanopterin (CH identical with H sub(4)MPT super(+)) and dihydrogen; CH sub(2) = H sub(4)MPT + H super(+) CH identical with H sub(4)MPT super(+) + H sub(2); Delta G degree ' = + 5.5 kJ/mol. In the following investigation, the formation of H sub(2), HD and D sub(2) was studied in experiments in which either the methylene group of CH sub(2) = H sub(4)MPT or water were deuterium labelled. In the case of CD sub(2) = H sub(4)MPT and H sub(2)O, the dihydrogen formed immediately after the start of the reaction was composed of approximately 50% HD and 50% of H sub(2) at all pH tested. In the case of CH sub(2) = H sub(4)MPT and D sub(2)O, the dihydrogen generated was composed of approximately 50% HD and 50% D sub(2) at pD 5.7 and of approximately 85% HD and 15% D sub(2) at pD 7.0. Evidence is presented that the enzyme catalyzes a CH identical with H sub(4)MPT super(+)-dependent isotopic exchange between HD and H sub(2)O and between HD and D sub(2)O, yielding H sub(2) and D sub(2), respectively. A catalytic mechanism aimed to explain these findings is discussed.