W0062

Structure of Formaldehyde Ferredoxin Oxidoreductase from Pyrococcus Furiosus. Yonglin Hu, Douglas C. Rees, Division of Chemistry and Chemical Engineering, 147-75CH California Institute of Technology, Pasadena, CA 91125, Roopali Roy, Michael W.W. Adams, Department of Biochemistry and Molecular Biology University of Georgia, Athens GA 30602

Formaldehyde ferredoxin oxidoreductase (FOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homotetramer that contains a tungsten-molybdopterin cofactor and an Fe₄S₄ cluster. The crystal structures of FOR and its complex with glutarate have been solved by molecular replacement using the structure of the homologous Pf AOR and refined to 1.85Å and 2.4Å, respectively. The FOR and the FOR-glutarate complex crystals are isomorphous and belong to space group P2₁2₁2₁, with cell dimensions of a=100 Å, b=170Å, and c=180Å, and one tetramer per asymmetric unit. The structure consists of three domains. A cavity at the interface between domains 2 and 3 leads from the tungsten atom to the surface of the protein. The tungsten-molybdopterin and the Fe₄S₄ cofactors are also located at the interfaces between the domains. The tungsten atom is near the center of the protein, and is coordinated by all the four sulfur atoms from the two molybdopterins. In the FOR-glutarate complex structure, the glutarate molecule is bound noncovalently in the cavity, with one carboxylate group positioned near the tungsten site, and the second one anchored to the protein by electrostatic interactions with the side chains of Arg 481 and Arg 492. Based upon a comparison of the FOR and AOR structures and the protein-glutarate interactions in FOR-glutarate complex, we propose that residues Thr 240, Glu 308, and His 437 may participate in the proton transfer coupled with electron transfer, whereas residue Tyr 416 may form a hydrogen bond with the -CHO group of the bound aldehyde substrate, positioning this group toward the tungsten atom during the catalytical process. The counterparts of these four highly conserved residues may play similar roles in other AOR family tungstoenzymes. Residues Arg 481 and Arg 492 might be responsible for the specificity of FOR by recognizing a negatively charged group of the substrate.