Structure and Function of the Methyl-coenzyme M Reductase Activation complex
Eduardus C. Duin (Principal Investigator)
Auburn University
William B. Whitman (Principal Investigator)
University of Georgia at Athens
Methanogenesis is a major component of the biogeochemical carbon cycle that maintains the chemical composition of the Earth’s atmosphere. Unfortunately, anthropogenic methane production has led to rapid increases in atmospheric methane, contributing up to 30% of the current global warming. The methyl-coenzyme M reductase (or Mcr) is a key enzyme in the methane cycle and essential for both the biological formation of methane by methanogenic archaea and the anaerobic oxidation of methane by the closely related methanotrophic archaea. This project addresses fundamental questions in the biochemistry of methane formation and consumption by these archaea. Essential for Mcr catalytic activity is a nickel-containing tetrapyrrole coenzyme F430, which must be in its most reduced form for the enzyme to be active. Many conditions can result in oxidation of the nickel and inactivation of the enzyme. Thus, the formation and stability of the reduced F430 are critical for maintaining enzyme activity.
The proposed research will seek to understand the mechanism of Mcr activation, i.e. conversion of the inactive Mcr to active enzyme. In previous results, evidence was obtained for a large complex that included subunits of Mcr as well as “methanogen marker proteins” known to be required for Mcr activation. Individual proteins will be tagged to allow for rapid purification of the complex from cell extracts of the methanogenic archaeon Methanococcus maripaludis. The complex will be characterized to determine its protein and coenzyme composition. Conditions will be developed to preserve or stabilize any labile components. Further experiments will be performed to optimize the activation of the recombinant Mcr expressed in M. maripaludis. Based upon recent progress isolating activated Mcr, work will be performed to shorten and standardize these methods. Key to these experiments include developing preincubation conditions for the cells prior to lysis and methods for rapid purification of recombinant Mcr. Lastly, a genetic test will be developed to determine whether or not recombinant Mcr from diverse sources are active in whole cells of M. maripaludis. These experiments will determine if the Mcr activation system is universal, i.e. capable of working with enzymes from very different methanogens and methanotrophs.
These approaches are interrelated. Characterization of the activation complex will elucidate its possible roles and have the practical consequence of developing the methods for production of reagent quantities of stable proteins for activation assays. Further studies to develop the methods for purification of activated Mcr will allow for detailed studies of its mechanism as well as the activation process. The survey of Mcr activation in whole cells will determine whether or not the activation process is highly conserved among methanogens and methanotrophs. These studies will enable a high level of control of these processes, which would greatly benefit core research areas of the Department of Energy's Office of Science and help achieve the planned 30 % reduction in methane emissions. For instance, the proposed research will enable the design of specific methanogenesis inhibitors for use in agriculture. It could also lead to the design of a Mcr mimic, ie. a chemical catalyst that could efficiently oxidize methane for production of high value products. Methane is also a common fuel, and biological methane production catalyzed by methanogens has great potential as an alternative fuel source.
