Methane and ethane production rates by methyl-coenzyme M reductase in cell extracts from different methanogens

Abstract

Methyl-Coenzyme M reductase (Mcr) plays an important role in the regulation of the global carbon cycle. The enzyme catalyzes the reversible conversion of methyl-coenzyme M and coenzyme B to methane and the corresponding heterodisulfide CoM-S-S-CoB, which constitutes the first step of the anaerobic oxidation of methane. Mcr homologs of varies methanogenic archaea also catalyze the anaerobic oxidation of extended alkanes. Fully active, purified enzyme is exclusively achievableviastrictly anaerobic purification fromMethanothermobacter marburgensis. This microbe expresses two isoenzymes and most studies were performed with isoenzyme I that exhibits a limited substrate-promiscuity (c.a. 0.5%) towards the homologous substrate ethyl-coenzyme M. Here, cell-free lysates from the different species such asMethanosarcina mazei, Methanococcus maripaludis, Methanothermococcus okinawansis, andMt. marburgensiswere screened for Mcr activity and substrate promiscuity. An assay relying on titanium (III) citrate and cobalamin to regenerate coenzyme B and coenzyme M was used to test the ability of different cell extracts for the catalytic activity towards the C2-substrate ethyl-coenzyme M. Cell extracts fromM. mazeishowed a ratio of ethane-to methane-production of ca. 8% at 37 °C, and about 14% at 49 °C. The level of substrate-promiscuity towards ethyl-coenzyme M forM. marburgensiscell extracts under our assay conditions were much higher than previously reported for purified isoenzyme I, indicating that isoenzyme II is much more promiscuous for ethane formation than isoenzyme I. Our experiments demonstrate that Mcr activity can be quickly and conveniently studiedviacell-free lysates, and that substrate promiscuity towards ethane formation is generally larger than anticipated.