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

The enzyme methyl-coenzyme M reductase (Mcr) is responsible for most of the biologically produced methane. Mcr catalyzes the reversible conversion of methyl-coenzyme M and coenzyme B to methane and the corresponding heterodisulfide CoM-S-S-CoB as the last step in all methanogens. In anaerobic methanotrophs, it catalyzes the same reaction to proceed in the reverse direction. While Mcr has been historically considered as an enzyme exclusive to the one-carbon metabolism, recent studies have demonstrated that homologs of Mcr have evolved to catalyze the first step of the archaeal anaerobic oxidation of medium and long-chain alkanes to their corresponding alkyl-coenzyme M thioethers. While ample metagenomic studies and data related to Mcr is available, in vitro experiments are limited, because purifying Mcr in its active nickel-I form is challenging. Fully active enzyme has only been reported for Methanothermobacter marburgensis isoenzyme I, from which most of the enzyme studies have been performed. To get an overview of the substrate scope of different Mcr variants, we tested cell-free lysates from the methanogens Methanosarcina mazei, Methanosarcina acetivorans, Methanococcus maripaludis, Methanothermococcus okinawensis, and M. marburgensis for their rates to convert methyl- and ethyl-coenzyme M to methane and to ethane, respectively. Cell extracts from M. mazei showed an ethane production rate of ca. 8% relative to methane production at 37 {degrees}C, and about 14% at 49 {degrees}C in an assay relying on titanium (III) citrate and cobalamin to regenerate coenzyme B from the CoM-S-S-CoB heterodisulfide. For cell extracts of M. marburgensis, we found an ethane-to-methane production rate of 8%, which is substantially higher than the reported value of ca. 0.3% for the ratio of their maximal catalytic rate for purified isoenzyme I. Since M. marburgensis is known to express two isoenzymes depending on the growth conditions, we hypothesize that isoenzyme II is substantially more promiscuous towards ethane formation than the well-described isoenzyme I. Which it is still challenging to obtain accurate kinetic parameters of the Mcr-catalyzed reaction, our experiments demonstrate that Mcr activity can be quickly and conveniently studied via cell-free lysates, and that substrate promiscuity towards ethane formation is generally larger than anticipated.