An Expanded Molecular Model for the Activation of Methyl-Coenzyme M Reductase.

Methyl-coenzyme M reductase (Mcr) catalyzes the terminal carbon reducing step in the methanogenesis cycle and has been closely scrutinized since its discovery nearly five decades ago. One critical gap in our knowledge is the structure of the protein complex necessary for the reductive activation of the nickel atom coordinated within the Mcr coenzyme F₄₃₀. Phylogenomic analysis previously identified 17 genes of unknown function that were found only in the genomes of sequenced methanogens and encode so-called "methanogenesis marker proteins" (Mmp1 through Mmp17). The functions of most Mmps remain largely unknown. Here we describe a complex formed from methanogenesis marker proteins 3, 5, 6, 7, 15, 17, AtwA, McrC, and two proteins with domains of unknown function (DUF2098 and DUF2111). Expression of the operon encoding these mmp genes from Methanosarcina acetivorans in Escherichia coli resulted in the formation of a large iron-sulfur cluster containing protein complex. Subsequent structural modeling revealed a putative complex comprised of a dimer of heterodecamers containing a total of ten [8Fe-9S-C] clusters, four Mg²⁺-ATPs, three [4Fe-4S] clusters, two Zn²⁺ ions, and two Mg²⁺-FAD ligands that interact with two Mcr holoenzymes. Systematic individual overexpression of the components of the complex in a native host, Methanococcus maripaludis, with affinity chromatography pull-downs and analysis by tandem mass spectrometry revealed a native protein complex formed in agreement with the predicted structure. These results provide a more complete molecular model of the activation complex catalyzing the ATP-dependent reductive activation of Mcr.