Methanogens, which are found in nearly all anaerobic habitats, are key players in the global carbon cycle, producing 1 gigaton of methane gas annually. Methanogens survive by converting carbon in low-oxygen sediments to methane gas, which is then oxidized to carbon-dioxide in the aerobic environment, thereby playing a key role in the global carbon cycle. Recent preliminary evidence supports the involvement of a large multienzyme complex in biological methane production and resolves unexplained mutant phenotypes reported in the literature. Multienzyme redox complexes are important because cells must coordinate the relative ratio of oxidized vs reduced electron carriers with the catalytic requirements of enzymes involved in central metabolism. This project seeks to elucidate the basic biochemical principles by which this novel multi-enzyme complex coordinates metabolic processes in methanogens. Defining the mechanism by which this multi-enzyme complex functions will also help us understand the basic underlying biochemical principles related to multienzyme redox complexes in other archaea, bacteria, or eukaryotes. The project will support student training in anaerobic microbial physiology and biophysical biochemistry.
The multi-enzyme complex is comprised of three enzymes, CoM-S-S-CoB heterodisulfide reductase (HdrD), acetyl-CoA decarbonylase/synthase (ACDS), and methylene tetrahydromethanopterin reductase (Mer). The studies supported by this award will use genetics, biophysical biochemistry, and molecular biology techniques to define the mechanism by which the HdrD:ACDS:Mer complex integrates flux through electron transport and metabolism. The HdrD:ACDS:Mer multienzyme complex directs carbon to biosynthesis or methanogenesis dependent on the ratios and oxidation state of electron carriers in the cell. Specific aims for the study include verifying HdrD:ACDS:Mer complex formation by in vivo crosslinking, affinity purification, and mass spectrometry of all component subunits. Experiments will also be conducted to investigate redox-dependent crosstalk between enzymes in vivo. Acetyl-CoA production and methyl-tetrahydromethanopterin-dependent reduction of ferredoxin, F420, and CoM-S-S-CoB electron carriers will be measured in coupled reactions catalyzed by the HdrD:ACDS:Mer complex in mutant cell extracts by UV/Vis spectrophotometry. Results of the study will be disseminated through presentations at scientific conferences and through peer-reviewed publications.