Unique Biochemistry at the Interface of One- and Two-Carbon Metabolism in Methanogens and other Archaea

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This project is providing biochemical understanding of how a unique multienzyme complex, the acetyl-CoA decarbonylase/synthase (ACDS) complex, functions in methanogens and other species of Archaea. The ACDS complex catalyzes an unusual energy-yielding, metal-based decarbonylation reaction in which both the C-C and C-S bonds of the acetyl group in acetyl-CoA are broken, or formed. ACDS plays a central role in acetate decomposition to CO2 and methane by methanogens, with approximately two-thirds of the annual methane production on Earth being derived from this process. In methanogens consuming CO2 and hydrogen, ACDS acts in reverse to synthesize acetyl groups for carbon assimilation during growth. Thus, ACDS plays an important role in the global carbon cycle both in the release of carbon in the form of CO2 and methane, and by its involvement in the autotrophic fixation of CO2. Acetyl group synthesis and cleavage take place at an active site Ni-Ni- and Fe/S-containing metal center (the A cluster) bound to the protein beta subunit. There is considerable interest in understanding the unusual bioinorganic mechanism of this reaction, and this project is focused on analysis of the structure, spectroscopic, and biochemical properties of a newly isolated acetyl-nickel organometallic intermediate formed at the A cluster, critical to the mechanism of acetyl-CoA synthesis and cleavage. Collaborative X-ray crystallographic analyses are being carried out on the ACDS beta subunit in its acetylated form to provide structural information that will contribute new insight into how enzymes are able to exploit unusual organometallic chemistry for interconversion of one- and two-carbon substrates. Complementary biochemical and FTIR spectroscopic analyses are being applied to the ACDS beta subunit enzyme-acetyl intermediate to provide information defining the reactivity and functional properties of the A cluster acetyl-Ni species. In addition, this project is testing how steric effects generated by domain-specific conformational changes influence the coordination geometry and stability of the A cluster acetyl-Ni intermediate. New insight into the mechanism of acetyl C-C and C-S bond activation is being obtained from characterization of several domain-truncated and single site-directed mutants to determine how different protein conformational states enforce specific metal center coordination geometries to promote the stepwise interconversion of C-1 and C-2 intermediates.Broader Impacts This research is concerned with fundamental knowledge of the biochemistry and physiology of Archaea, organisms that constitute the third-domain of life. Archaea comprise as much as 20% of the total microbial biomass in the oceans and methanogens are found in especially high numbers in the digestive systems of ruminants, as well as being widely distributed in soil and aquatic environments. Methanogens present a direct advantage to society in their use for bioremediation and detoxification of anoxic wastes. In addition, methane produced as the end product of methanogenic metabolism is a clean fuel, and a better fundamental understanding of methane biochemistry has impact on societal efforts to develop alternative energy sources. Thus, the results from this work have a broad impact, not only for the useful exploitation of the metabolic potential of the Archaea for industrial and agricultural purposes, but also for a better understanding of the world's ecology and the environment. Important educational and teaching components of this project further extend its broader impact. Students at all levels (high school through postdoctoral) and technologists are actively engaged in characterizing oxygen-sensitive multi¬enzyme systems by state-of-the-art biochemical, spectroscopic, enzymatic, and molecular biological techniques, and the project embraces participation of underrepresented and disadvantaged students as well. Furthermore, in this research, new collaborations have been established that are broadening the interdisciplinary knowledge and skill sets of students and postdoctoral associates at several institutions. This contributes significantly to enhancement of infrastructure for research and education.