We propose to determine the chemical mechanism by which methanogens utilize H2 to reduce CO2 to CH4. We also wish to determine how this reaction is coupled to ATP production, our hypothesis being that CH4 production in an inner compartment of a methanogenic bacterium (e.g., Methanobacterium thermoautotrophicum) results in a local depletion of H+ and the production of a proton gradient which becomes the proximal driving force for phosphorylation of ADP. In the initial period of this grant we will examine these questions by approaching the followig experimental goals: (1) We will characterize the prosthetic groups and chemical mechanism of the uptake hydrogenase of M. thermoautotrophicum, which we have recently isolated; (2) We plan to isolate and characterize the formate dehydrogenase catalyzing the initial step of CO2 reduction; (3) We will also attempt to determine how methyl coenzyme M, the penultimate carbon compound in methane synthesis, is itself metabolized. In (1-3), we will isolate the enzymes, characterize their subunit, metal and organic cofactor composition, using methods of organic, inorganic and physical chemistry successfully applied to flavoproteins, nitrogenase, P-450 oxygenases, and B12 enzymes in our laboratories for over a decade. In parallel to these studies we will (4) use 13C NMR to study the intermediate patterns in 13C02 assimilation in whole cells in the probe of high field (270 and 500 MHz) spectrometers, in an attempt to deduce the chemistry of the intermediate steps; and (5) use 31p NMR to assess the pH gradients inside cells from the pH sensitive chemical shifts of phosphate ions in the medium and in intracellular compartments. We believe that these studies may help afford a comprehensive and accurate picture of the chemistry and physiology of methane biogenesis.
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