Characterization of Heterogeneous Nickel Sites in CO Dehydrogenases from Clostridium thermoaceticum and Rhodospirillum rubrum by Nickel L-Edge X-ray Spectroscopy C. Y. Ralston, ²,‡ Hongxin Wang, ‡ S. W. Ragsdale, § M. Kumar, § N. J. Spangler, | P. W. Ludden, | W. Gu, ‡ R. M. Jones, ⊥ D. S. Patil, ⊥ and S. P. Cramer* ,‡,⊥ Contribution from the Department of Applied Science, UniVersity of California, DaVis, California 95616, Department of Biochemistry, UniVersity of Nebraska, Lincoln, Nebraska 68583, Department of Biochemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706, and Lawrence Berkeley National Laboratory, Berkeley, California 94720 ReceiVed March 16, 2000 Abstract: Carbon monoxide dehydrogenase from Clostridium thermoaceticum (Ct-CODH) is a nickel-containing enzyme that catalyzes acetyl-CoA synthesis and CO oxidation at two separate Ni sites, the A-cluster and C-cluster, respectively. Carbon monoxide dehydrogenase from Rhodospirillum rubrum (Rr-CODH) contains only a C-type cluster and catalyzes only CO oxidation. We have used L-edge X-ray absorption spectroscopy to study the Ni electronic structure of these two enzymes. The spectra indicate that most of the Ni in as- isolated Ct-CODH is low-spin Ni(II). Upon CO treatment, a fraction of the nickel is converted either to high- spin Ni(II) and/or to Ni(I). Ni in dithionite-reduced Rr-CODH also exhibits a clear low spin Ni(II) component, again mixed with either high-spin Ni(II) or Ni(I). The spectrum of Rr-CODH shifts to higher energy upon indigo carmine oxidation, suggesting either that most of the high-spin Ni(II) is converted to low-spin Ni(II) and/or that some Ni is oxidized between these two forms. These results are discussed and compared with recent L-edge spectra for the Ni site in hydrogenase. Introduction The Wood-Ljungdahl pathway describes the biochemical steps involved in anaerobic fixation of carbon dioxide and synthesis of acetate. 1,2 Apart from their biochemical and environmental significance, these reactions are analogous to important industrial processes. 3 In Clostridium thermoaceticum, CO oxidation and acetyl-CoA synthesis are catalyzed by carbon monoxide dehydrogenase (Ct-CODH), a 310 kDa (R) 2 tet- ramer. 4,5 In the photosynthetic bacterium Rhodospirillum ru- brum, there is a related enzyme (Rr-CODH)sa monomer that catalyzes only the first reaction. Ct-CODH catalysis involves two physically distinct sites, 4-8 the “C-cluster” and “A-cluster”, which both contain Ni and Fe. 9-13 Each R dimer contains 2 Ni and 11-14 Fe 7,14 and presumably incorporates one A-cluster and one C-cluster. There is also a conventional [Fe 4 S 4 ] 2+/1+ “B-cluster”, 15 which transfers electrons between the C-cluster and external redox agents. 5 The A-cluster contains the acetyl-CoA synthesis site and is EPR-silent in the as-isolated “A ox ” form. It generates a “Ni- Fe-C” EPR signal when Ct-CODH is treated with CO 16,17 to yield “A red -CO”. Mo ¨ssbauer and ENDOR studies suggest a structure involving Ni bridged to an Fe 4 S 4 cluster. 8,11,18,19 The CO oxidation site C-cluster has been proposed to have a similar structure. 13 This cluster occurs in two EPR-active forms, “C red1 ” and “C red2 ”, as well as an EPR-silent “C ox ” form, along with other states. Rr-CODH is a 66.9-kDa protein containing 1 Ni and 7-8 Fe, 20 with an associated 22-kDa subunit containing an additional ² Current address: Department of Biophysics and Physiology, Albert Einstein College of Medicine, Yeshiva University, The Bronx, NY 10461. ‡ University of California. § University of Nebraska. | University of Wisconsin. ⊥ Lawrence Berkeley National Laboratory. (1) Ferry, J. G. Annu. ReV. Microbiol. 1995, 49, 305-333. (2) Ragsdale, S. W.; Kumar, M. Chem. ReV. 1996, 96, 2515-2539. (3) Qiu, D.; Kumar, M.; Ragsdale, S. W.; Spiro, T. G. Science 1994, 264, 817-819. (4) Raybuck, S. A.; Bastian, N. R.; Orme-Johnson, W. H.; Walsh, C. T. Biochemistry 1988, 27, 7698-7702. (5) Kumar, M.; Lu, W.-P.; Liu, L. F.; Ragsdale, S. W. J. Am. Chem. Soc. 1993, 115, 11646-11647. (6) Shin, W.; Lindahl, P. A. J. Am. Chem. Soc. 1992, 114, 9718-9719. (7) Xia, J. Q.; Sinclair, J. F.; Baldwin, T. O.; Lindahl, P. A. Biochemistry 1996, 35, 1965-1971. (8) Xia, J.; Lindahl, P. A. J. Am. Chem. 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W.; Hoffman, B. M. Biochemistry 1991, 30, 431-435. (19) Xia, J. Q.; Hu, Z. G.; Popescu, C. V.; Lindahl, P. A.; Mu ¨nck, E. J. Am. Chem. Soc. 1997, 119, 8301-8312. CO + H 2 O f CO 2 + 2H + + 2e - CH 3 -THF + CoA‚SH + CO f CoA‚SCOCH 3 + 2THF 10553 J. Am. Chem. Soc. 2000, 122, 10553-10560 10.1021/ja0009469 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/11/2000