Abstract 1633 Investigation and characterization of the activation complex, A3a, of methyl coenzyme M reductase

Methane is a potent greenhouse gas formed geologically in anaerobic environments from the thermal decomposition of kerogen (fossil fuel), from the reduction of oxidized carbon compounds by archaeal methanogens, and possibly by abiotic synthesis. Anaerobic microbial methane metabolism is one key part of the global carbon cycle, which controls the methane emission to the atmosphere. Methanogenic archaea and anaerobic methanotrophic archaea (ANME) in anoxic environments are the major groups involved in microbial methane metabolism. Methyl-Coenzyme M reductase (Mcr) catalyzes a reversible reaction that involves the reduction of methyl-coenzyme M (CH3-S-CoM) by coenzyme B (HS-CoB) to generate methane, CH4 and the heterodisulfide, CoM-S-S-CoB. Therefore, it is the key enzyme in both the anaerobic oxidation of methane by ANaerobic MEthane oxidizers (ANME) and production of methane by methanogens, using any of the three known pathways: hydrogenotrophic, methylotrophic and acetoclastic. Mcr contains two coenzyme F430 as its cofactor, a nickel-containing tetrapyrrole, in its active site. Three different oxidation states of the nickel are accessible. The Mcr-red1 form, with an oxidation state of +1, is the active, reduced form of this enzyme. The Mcr-ox1 form, with an oxidation state of +3, is the inactive ready form but still EPR active, while the inactive, EPR silent form (Mcr-silent), has an oxidation state of +2. No chemical method is available to reduce Mcr-silent to Mcr-red1 in vitro. Leveraging on the reduction of Mcr silent in-vivo by A3a, an activation complex first isolated by Ralph Wolfe and his co-workers, but remaining uncharacterized, our research is aimed at isolating this complex from Methanothermobacter marburgensis. Here we will present the purification and characterization of the large A3a complex.