Methyltransfer reactions in the reductive acetyl-Coenzym A pathway

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Anaerobic bacteria and archaea use homologous enzymes to convert CO2 and H2 to acetyl-CoA and methane, which determine since the dawn of life the global carbon cycle on earth. In the last funding period, we focused on three proteins involved in methyl transfer in the reductive acetyl-CoA pathway. We could show that orf7 of the gene cluster of the acetyl-CoA pathway encodes the reductive activator of the central B12-dependent methyl group acceptor and donor CoFeSP. To achieve activation, the activator catalyzes an ATP-dependent intermolecular electron transfer against a redox potential gradient between a [2Fe2S] cluster and a Co(II) ion. The structure of the complex between activator and CoFeSP together with spectroscopic and kinetic studies have demonstrated a novel way of how an activator alters the redox potential of the cofactor in the target protein to render electron transfer favorable. For CoFeSP, we have developed a FRET assay and combined it with PELDOR measurements now allowing to determine the magnitude of the conformational changes (PELDOR spectroscopy) together with the kinetics of the change (stopped-flow kinetics FRET). The final reaction in acetyl-CoA formation is catalyzed by acetyl-CoA synthase, whose unstable Ni-Ni-[4Fe4S] center we could analyze with a resolution (dmin = 1.4 Å) using a truncated construct.In the next funding period, we want to clarify (I) the mechanism of the reductive activator such as the function and mechanism of ATP hydrolysis and the role of dissociation of the CoFeSP-activator complex for electron transfer. The rate-determining step of the reaction needs to be determined, as well as the position of the electron-donating [2Fe2S] cluster domain in the complex. (II) Where the substrates and intermediates CO, CH3CO+, CH3+ and coenzyme A bind on the Ni-Ni-[4Fe4S] cluster of acetyl-CoA synthase is still unknown and we want to use the good quality of our crystals to gain a first look into the substrate binding place and mechanism. (III) We want to investigate the homologous enzymes from methanogenic archaea that, in contrast to the bacterial proteins, form an approximately 2.5 MDa complex, containing acetyl-CoA synthase, carbon monoxide dehydrogenase and CoFeSP. The reactions of the three enzymes are coordinated in the complex in an as yet unknown way. While bacterial CoFeSP is methylated by methyltetrahydrofolate-dependent methyltransferase, the CoFeSP homologs from methanogens can methylate itself using CH3-methanopterin as substrate. Comparing the enzymes of bacteria and archaea will give further insight into the early stages in the evolution of B12-dependent methyltransferases and the origin of methanogenesis and acetogenesis.