Methyl-coenzyme M reductase (MCR) plays an important role in global methane cycle, which is common to all methanogenic pathways and catalyzes the terminal step of methane formation. Homologs of MCR are also abundant in the anaerobic methanotrophic archaea (ANME) and short-chain alkane-oxidizing archaea (ANKA), involved in the anaerobic oxidation to CO2 of alkanes such as methane, butane, propane, and ethane. The biological mechanisms controlling anaerobic oxidation of alkanes have not been reported due to an inability to obtain pure culture and extremely long doubling times of ANME/ANKA enrichment cultures. Although extensive studies on methanogenic MCR have revealed its catalytic properties and mechanism insights, the knowledge about its assembly, maturation and activation is still limited. The study of heterologous expression of methanogenic and ANME/ANKA MCR is not only necessary to elucidate the biochemical characteristics of MCR from uncultivated organisms but also provides new insights for catalytic and mechanistic studies of MCR homologs. A robust expression system was developed for overexpression and purification of recombinant MCR in Methanococcus maripaludis, a model methanogen with well-developed genetic tools. Methanogenic and ANME MCRs are successfully expressed and assembled in M. maripaludis. Using this expression system, we revealed the important roles of accessory proteins (McrC and McrD) in MCR assembly. The accessory proteins are not required in one operon for MCR assembly, but they could function in trans for MCR assembly and this assistance is organism-specific. Moreover, McrG is shown to be a compatible subunit which could assemble into MCR complexes from distinct origins. McrG may bind F430 since the free McrG containing F430 dissociated from the holo-MCR after storage at -20°C. However, this hypothesis is still under investigation. Taken together, a possible assembly order was proposed that McrA and B forms an initial complex, and McrG is the last subunit joined the McrAB complex bringing the coenzyme F430. The presence of the accessory proteins assists the heterologous production of holo-MCR during assembly.