This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nickel- and iron-containing carbon monoxide dehydrogenases play important roles in the carbon and energy metabolism of a wide range of anaerobic bacteria. In addition, all of the methanogens and certain other archaeal species such as Archaeoglobus also rely on CO dehydrogenase for critical steps needed for carbon assimilation under a variety of growth conditions. When compared with its closest bacterial relatives, however, the major CO dehydrogenase present in methanogens and other Archaea is unique, both structurally and biochemically. Crystallographic structures have been obtained for two different types of Ni- and Fe-containing CO dehydrogenases from bacteria (1-4). The methanogen enzyme represents a third major form of CO dehydrogenase, and although significant biochemical information exists, no detailed crystallographic structure is yet available. In methanogens, CO dehydrogenase is present as an alpha2epsilon2 heterotetramer of 220 kDa, in which the 90 kDa alpha subunits show some regions of homology to bacterial counterparts, but also deviate markedly -- containing substantial stretches that are unrelated, including sequences that encode two additional Fe4S4 clusters (designated FS1 and FS2) in each alpha subunit, not found in bacteria. The 20 kDa epsilon subunit has no significant homologs in bacteria, and is considered one of the so-called archaeal signature proteins (5). In methanogens, the a2e2 CO dehydrogenase is tightly associated with three other types of subunits that include acetyl-CoA synthase (beta), and a corrinoid protein subcomponent (containing gamma and delta subunits). Together these five subunits combine to form a multienzyme complex (designated ACDS) of approximately 2,000 kDa, with eight copies of each subunit in an oligomeric arrangement unique to methanogens and Archaeoglobus. Spectroscopic data indicate that the alpha2epsilon2 protein contains an active site Ni- and Fe-containing C` cluster, at which CO is oxidized to CO2, whose structure is likely similar to that in bacterial CODHs. Although, uncertainty still exists about that cluster -- i.e., the number of inorganic sulfides is unclear (four or five?), and different coordination environments for Ni and one of the Fe atoms were reported in different crystallographic studies. The `B` cluster, which is more similar to a typical ferredoxin Fe4S4 center, is present in all CODHs with an identifiable 4-cysteine motif. However, the `D` cluster cysteine ligands in the bacterial proteins are remarkably absent in the archaeal a2e2 protein. The spatial arrangement of C, B, and D clusters, determined from the bacterial structures, indicates that the electrons released from CO oxidation are funnelled in each subunit from the C centers to B centers, and merge at a single D center formed between the subunits. In contrast, the D center cysteines are not present in the methanogen CODH alpha subunit sequences. Indeed, two additional 4Fe centers (FS1 and FS2) are present (as mentioned above), which indicates that the pathway for electron flow may be significantly different in the methanogen enzyme. How it differs, along with information needed to trace the actual intraprotein electron transfer pathway, could be determined from a crystallographic structure of the a2e2 enzyme. It is not possible to model the structure of the methanogen protein based on available data because of extensive sequence differences. For example, in the acetogen CODH structure, a substantial part of an extended tunnel used to channel CO runs through a large N-terminal domain (about 320 amino acids that make up roughly 45% of the acetyl-CoA synthase subunit) that interfaces with CO dehydrogenase. That N-terminal acetyl-CoA synthase domain is completely absent in methanogens. Instead, a unique C-terminal region of the ACDS beta subunit exists (not found in bacteria) that we previously showed is essential for functional subunit-subunit interactions in the ACDS complex (6-9). The upshot is that the structural basis for CO channeling is also likely to be substantially different in the archaeal complex, which, again would be addressed ideally by crystallographic studies. Moreover, a crystallographic structure of the methanogen alpha2epsilon2 CO dehydrogenase would be a major contribution because it would allow a variety of additional structure-function comparisons to be made between the existing CODH structures and this third major form of the enzyme. Recently, we made important technical advances in the purification and crystallization of the alpha2epsilon2 CODH protein that gives good looking (optically clear, without steps or visible inclusons) brown, hexagonal rod-shaped crystals (approx. 0.5 mm x 0.1 mm diameter) for which we seek high resolution diffraction data. We have on-hand, for the experiments, several cryopreserved crystals, loop-mounted and cooled under different cryoprotectant conditions. References: 1. Dobbek, H., Svetlitchnyi, V., Gremer, L., Huber, R., and Meyer, O. (2001) Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster. Science 293, 1281-1285. 2. Drennan, C.L., Heo, J., Sintchak, M.D., Schreiter, E., and Ludden, P.W. (2001) Life on carbon monoxide: X-ray structure of Rhodospirillum rubrumNi-Fe-S carbon monoxide dehydrogenase. Proc. Natl. Acad. Sci. 98, 11973-11978. 3. Doukov, T.I., Iverson, T.M., Seravalli, J., Ragsdale, S.W., and Drennan, C.L. (2002) A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Science 298, 567-572 4. Darnault, C., Volbeda, A., Kim, E.J., Legrand, P., Vernede, X., Lindahl, P.A., and Fontecilla-Camps, J.C. (2003) Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase. Nature Struct. Biol. 10, 271-279 5. Graham, D.E., Overbeek, R., Olsen, G.J., amd Woese, C.R. (2000) An archaeal genomic signature. Proc. Natl. Acad. Sci. 97, 3304-3308. 6. Grahame, D.A., and DeMoll, E. (1996) Partial reactions catalyzed by protein components of the acetyl-CoA decarbonylase synthase enzyme complex from Methanosarcina barkeri. J. Biol. Chem. 271, 8352-8358. 7. Kocsis, E., Kessel, M., DeMoll, E., and Grahame, D.A. (1999) Structure of the Ni/Fe-S protein subcomponent of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila at 26 Angstrom resolution. Journal of Structural Biology 128, 165-174. 8. Gencic, S., and Grahame, D.A. (2003) Nickel in subunit beta of the acetyl-CoA decarbonylase/synthase multienzyme complex in methanogens: Catalytic properties and evidence for a binuclear Ni-Ni site. J. Biol. Chem. 278, 6101-6110.
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