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43766-G3
Production and Anaerobic Oxidation of Methane by Methanogenic Archea
Methyl-coenzyme M reductase (MCR) is the key enzyme in two important biological processes, the production of methane in methanogenesis and the activation of methane in the anaerobic methane oxidation:
CH3-S-CoM + HS-CoB ↔ CH4 + CoM-S-S-CoB
with: CH3-S-CoM, methyl coenzyme M; HS-CoB, coenzyme B; CoM-S-S-CoB, heterodisulfide of coenzyme M and coenzyme B. It has been proposed that anaerobic methane oxidation is a complete reversal of methanogenesis. To overcome the positive free-energy change the ‘reverse' pathway is coupled to sulfate reduction. Present in the active site of MCR is a nickel-containing tetrapyrrole (factor 430) that plays a central role in the catalysis.
The goal of our research is to elucidate the reversible reaction mechanism of MCR and the ways the activity of the enzyme is regulated in the cell. This will provide valuable information about the one-step activation of methane at ambient temperature and pressure that will be used to design a nickel-based catalyst that can perform this function. Secondly, the data should result in the development of inhibitors that prevent the production of methane in, for example, livestock and rice fields, which are the main contributors in the increase of atmospheric methane levels that in turn contributes to the greenhouse effect.
The PRF funding allowed us to investigate the interaction of several substrate analogs and inhibitors with MCR isolated from Methanothermobacter marburgensis. A highly exciting result was the discovery that incubation of MCR with bromomethane resulted in the formation of an organometallic methyl-nickel species in the active site of MCR. The evidence for this was provided by using carbon-13-labeled bromomethane. Studies with electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies showed the presence of a carbon-13 hyperfine coupling of 18-44 MHz. This large value showed unquestionably that the methyl group from bromomethane is directly coordinated to the nickel ion. This finding is important since several catalytic mechanisms have been proposed for MCR. The main difference being the first step: When methyl-coenzyme M approaches the nickel of factor 430 it can either transfer the methyl group forming a nickel-methyl species, or it binds via its thiol sulfur forming a nickel-thiol bond. Our finding does not prove, but is in line with the first type of mechanism. However, since we now know the spectroscopic properties of the nickel-methyl species it will allow easy detection of such a species in kinetic experiments with MCR. In addition it supports our DFT based theoretical reaction mechanism that includes such an intermediate. The EPR and ENDOR data have been published in the Journal of the American Chemical Society. The DFT paper will be submitted in the Fall of 2007.
The methyl-nickel species present in MCR shows some very interesting properties. Incubation of this form with coenzyme M resulted in the formation of methyl-coenzyme M. This represents a possible mechanism for the reverse reaction. Studies are planned for the interaction of this species with the heterodisulfide of coenzyme M and coenzyme B.
Incubation of MCR with another compound, 2-bromoethane sulfonate, resulted in the conversion of the nickel of factor 430 into an EPR-silent species. At the same time a stable organic radical is induced that is EPR active. X-ray absorption experiments are underway to investigate the nickel geometry in this state. Parallel ENDOR measurements should help with the characterization of the radical species. Although it is generally expected that MCR uses a radical-type mechanism for the conversion of MCR, this is the first indication that a radical can be stabilized in the active site of MCR. Full characterization will be important for understanding the working of this enzyme.
We would like to thank the PRF for their support. The Department of Chemistry and Biochemistry provided the salaries for the two students working on this project, Ms. Na Yang, and Mr. Mi Wang. The PRF grant was used to buy the necessary supplies and chemicals.
