|dc.description.abstract||To use methane as a biocatalyst, it is important to establish a cell free system to convert the inactive form of methane forming enzyme i.e Methyl-coenzyme M reductase (MCR) to the active form. MCR catalyzes the reversible reduction of methyl-coenzymeM (CH3-S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide CoM-S-S-CoB (HDS). It contains the hydroporphinoid nickel complex coenzyme F430 in its active site, and the Ni center has to be in its Ni(I) valence state for the enzyme to be active. Until now, no in vitro method that fully converted the inactive MCRsilent-Ni(II) form to the active MCRred1-Ni(I) form has been described. With the potential use of recombinant MCR in the production of biofuels and the need to better understand this enzyme and its activation process, we studied its activation under nonturnover conditions and achieved full MCR activation in the presence of dithiothreitol and protein components A2, an ATP carrier, and A3a. It was found that the presence of HDS promotes the inactivation of MCRred1, which makes it essential that the activation process is isolated from the methane formation assay, which tends to result in minimal activation rates. Component A3a is a multienzyme complex that includes the mcrC gene product, an Fe-protein homolog, an iron-sulfur flavoprotein, CODH/ACS complex and protein components involved in electron bifurcation. According to our postulated model of activation, electrons from polyferredoxin or an artificial electron source with a potential close to that of polyferredoxin (Eo' ~ 500 mV at pH 7 vs. SHE) can reduce the Fe-S cluster in PISF and concomitant hydrolysis of ATP by the A2 protein/Fe protein homolog will lower the potential of this cluster allowing reduction of the Ni centers on MCR. Activation studies were performed in the presence of the artificial electron sources dithionite (Eo' ~ 420 mV at pH 7 vs. SHE) or Ti (III) citrate (Eo' ~ 500 mV at pH 7 vs. SHE). The rate of methane formation increased almost two fold in the presence of McrC, PISF, the Fe-protein homolog, the A2 protein, and Ti(III) citrate as an artificial electron source. There was no activation observed when dithionite was used instead of Ti(III) citrate. The fact that activation is only observed in the presence of Ti(III)citrate which has a midpoint potential close to that of polyferredoxin (Eo' ~ 500 mV at pH 7 vs. SHE), would be in line with our proposal that polyferredoxin is the direct electron donor and possible A3a protein would be McrC, Iron Sufur Flavoprotein and Fe-Protein Homolog.
On the other hand, Methane is an important greenhouse gas and it has a global warming potential 21 times more than that of carbon dioxide. Digestive processes of ruminants contribute a significant amount of methane. It has been estimated that cattle alone are responsible for emission of around 11-17% of methane globally. Additionally, between 2-12% of ingested gross energy of ruminants is lost due to the formation of methane. This loss of energy could be potentially used by the animal. Hence, controlling methane formation is important from the perspective of both environmental impact and animal productivity. 3-Nitrooxypropanol (3-NOP) has been identified and shown to be effective at inhibiting methane production both in vitro and in vivo with no signs of animal toxicity. 3-NOP is speculated to inhibit the key enzyme of methanogenesis, i.e. MCR, however, no studies describing the effect of 3-NOP against MCR have been reported in the literature to date. Considering this fact effect of 3-NOP on MCR was studied and it was found that 3-NOP quenches the active form of MCR via a radical type mechanism in which nitrite is released as a byproduct of this reaction.||en_US