Molybdenum Sites of Sulfite Oxidase and Xanthine Dehydrogenase. A Comparison by EXAFS

The molybdenum enzymes sulfite oxidase and xanthine dehydrogenase have been investigated by fluorescence-detected EXAFS using synchrotron radiation, and the results have been interpreted with improved EXAFS analysis procedures. A new treatment of EXAFS amplitudes has been developed which allows the extraction of meaningful Debye-Waller factors using experimentally derived functions. A search profile procedure has also been developed to aid in the treatment of minor EXAFS components. These methods have been used for a more detailed analysis of sulfite oxidase molybdenum EXAFS. They have also been used for analysis of the EXAFS of intact and cyanolyzed xanthine dehydrogenase, in both oxidized and reduced forms. Although the results are in qualitative agreement with recent work by Bordas et al., some significant quantitative differences are found. For oxidized sulfite oxidase, the analysis revealed two oxygens at 1.68 A and two or three sulfurs at 2.41 A, changing to one oxygen at 1.69 8, and three sulfurs at 2.38 A upon reduction. For oxidized, intact xanthine dehydrogenase the prediction was one oxygen at 1.70 A, one sulfur at 2.15 A, and two sulfurs at 2.47 A, changing to one oxygen at 1.68 8, and three sulfurs at 2.38 A upon reduction. Finally, in cyanolyzed xanthine dehydrogenase, two oxygens at 1.67 8, and two sulfurs at 2.46 A were found, which upon reduction changed to one oxygen at 1.66 A and two or three sulfurs at 2.33 A. In all cases there may be extra ligands which complete the molybdenum coordination sphere but contribute only weakly to the EXAFS. Molybdenum enzymes exhibit a broad range of chemical and spectroscopic behavior,’ and comprehending the structural basis for this diversity is a prerequisite for understanding and synthetically modeling their catalytic mechanisms. Previous results from X-ray absorption spectroscopy have suggested that the Mo sites of these enzymes can be classified as either “cluster” type such as in nitrogenase2-‘ or “oxo” t pe such as contained in xanthine Specifically, the EXAFS studies of nitrogenase have shown that the molybdenum is present in an Fe,Mo,S cluster, whereas the other Mo enzymes appear to have a Mo site with both sulfur donor and terminal oxo ligands. The structural differences between these two types of sites may be related to the fact that nitrogenase possesses an iron-molybdenum cofactor (“FeMo-co”),’O whereas the other enzymes cited contain a molybdenum cofactor (“Moa”) which is free of iron but contains a novel pterin component.” Even within the class of oxo-type Mo proteins there are substantial differences in properties. Xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase all have relatively low Mo redox potentialsI2 and a unique “cyanolyzable” sulfur.’ Upon treatment of these enzymes with cyanide, SCNis released and “desulfo” Mo proteins are formed which have no catalytic activity and even lower redox potentials. A molybdenum protein from Desulfovibrio gigas has properties similar to these desulfo proteins.” In contrast, sulfite oxidaseI4 and nitrate red~ctase’~ have substantially higher Mo redox potentials, and although they are inhibited by cyanide in their reduced states, this process is re* To whom correspondence should be addressed at the Exxon Research and sulfite oxidase, P and nitrate r e d ~ c t a s e . ~ Engineering Co. versible by oxidation and no SCNis released.16 Thus, a distinction can be made between low potential and high potential (1) For a recent review of molybdenum biochemistry, see: “Molybdenum and Molybdenum-Containing Enzymes”, M. P. Coughlan, Ed., Pergamon Press: New York, 1980. (2) S. P. Cramer, K. 0. Hodgson, W. 0. Gillum, and L. E. Mortenson, J . Am. Chem. Soc., 100, 3398-3407 (1978). (3) S. P. Cramer, W. 0. Gillum, K. 0. Hodgson, L. E. Mortenson, E. I. Stiefel, J. R. Chisnell, W. J. Brill, and V. K. Shah, J . Am. Chem. Soc., 100, ( 4 ) T. E. Wolff, J. M. Berg, C. Warrick, K. 0. Hodgson, and R. H. Holm, J. Am. Chem. SOC., 100, 4630-4632 (1978). (5) T. D. Tullius, D. M. Kurtz, Jr., S. D. Conradson, and K. 0. Hodgson, J . Am. Chem. Soc., 101, 2776-2779 (1979). (6) J. Bordas, R. C. Bray, C. D. Garner, S. Gutteridge, and S. S. Hasnain, J . Inorg. Biochem., 11, 181-186 (1979). (7) J. Bordas, R. C. Bray, C. D. Garner, S. Gutteridge, and S. S. Hasnain, Biochem. J. 191,499-508 (1980). (8) S. P. Cramer, H. B. Gray, and K. V. Rajagopalan, J . Am. Chem. Soc., (9) S. P. Cramer, L. E. Solomonson, M. W. W. Adams, and L. E. Mortenson, submitted to Biochem. Biophys. Res. Commun. (10) P. T. Pienkos, V. K. Shah, and W. J. Brill in “Molybdenum and Molybdenum-Containing Enzymes”, M. P. Coughlan, Ed., Pergamon Press, New York, 1980, pp 385-401. (1 1) J. L. Johnson in “Molybdenum and Molybdenum-Containing Enzymes”, M. P. Coughlan, Ed., Pergamon Press, New York, 1980, pp 345-383. (12) M. J. Barber, M. P. Coughlan, M. Kanda, and K. V. Rajagopalan, Arch. Biochem. Biophys., 201, 468-475 (1980). (13) J. J. G. Moura, A. V. Xavier, R. Cammack, D. 0. Hall, M. Bruschi, and J. LeGall, Biochem. J., 173,419-425 (1978). (14) S. P. Cramer, H. B. Gray, N. S. Scott, M. Barber, and K. V. Rajagopalan in “Molybdenum Chemistry of Biological Significances”, W. E. Newton and S. Osaka, Eds., Plenum Press, New York, 1980, pp 157-168. 3814-3819 (1978). 101, 2772-2774 (1979). 0002-7863/8l/l503-7721$01.25/0