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1 pp. -O Mo Mo Mo EC EC xanthine oxidoreductase XOR EC AO EC Structure and mechanism of molybdenum hydroxylase Ken Okamoto and Takeshi Nishino Department of Biochemistry, Nippon Medical School, Sendagi, Bunkyo- Ku, Tokyo, Japan A A XOR AO B B DMSO

2 A B XOR XOR B XOR Fe- SFAD Beinert ESR electron spin resonance Bray ESR XOR XOR XOR P XOR AO XOR NAD xanthine dehydrogenase XDH xanthine oxidase XO XO NAD O XO

3 XOR XOR XOR FAD nm nm Mössbauer circular dichroism ESR ESR Mo rapid slow rapid proton hyperfine splitting type type Rhodobacter capsulatus XOR -S- S O OH S O Desulfovibrio. gigas AO S - -pyridin- -yl- H- triazol- -yl pyridine- -carbonitrile FYX- Mo S Mo-SH magnetic circular dichroism X-ray absorption spectroscopy XOR A B O XOR XOR XOR XDH XO XOR kda A Fe- S FAD B Fe S I Fe S II Fe S II Fe S I Fe S II FAD NAD Fe S I FAD Fe S II FAD C Fe S II Fe S II electron sink FAD

4 A B C XOR TEI- Y- FYX- Ki M FYX- Arg Thr Glu C Phe Phe edge-on interaction Glu Glu Glu Arg XOR Glu Arg Aspergillus nidulans, R. capsulatus XDH XOR Mo O -OH S -S- O

5 O Mo O Mo-OH DMSO O Howes Mo-C D. gigas AO Mo-O Mo-O-C H Mo S Mo S Mo-OH O ENDOR electron nuclear double resonance FYX-A Mo -OH-FYX- FYX- FYX- C B Mo-C Mo-O O-C B Mo-OH Glu Glu Mo-OH Mo-O- Mo-O-C Glu XOR AO R. capsulatus Glu Glu Glu Glu A FYX- -OH-FYX- B XOR-FYX- fo-fc map σ C XOR-FYX-

6 FYX- N C Mo-O Mo S Mo S Mo-SH Mo -OH FYX- XOR A C N Mo XOR N B FYX- N -OH Glu Glu Arg B AO Glu Arg Truglio R. capsulatus XOR XOR Glu Arg Phe Phe Glu C Glu ArgGlu Glu A B XOR- fo-fc map σ

7 Glu ph Glu kcat Arg Arg EV kcat A. nidulans Arg -hydroxypurine -hydroxypurine A B C AO

8 Arg Pauff R. capsulatus Arg -hydroxy- -methylpurine -O Arg Glu Glu Arg C Arg Glu N Glu Aa -OH Glu-O C Ab Glu N C Mo-O-C C H S S -SH Ac Ad -hydroxy- -methylpurine Mo Mo A C Arg B Arg Glu -hydroxy- -methylpurine Arg N Glu Pauff Glu AO AO Glu Glu Arg C O -O -OH Glu Stiefel, E.I.in Metal Ions in Biological System Sigel, A.&Sigel,H.eds., pp., Marcel Dekker, New York. Hille, R.Trends Biochem. Sci.,,. Hille, R.Chem. Rev.,,. Mendel, R.R.J. Exp. Bot.,,. Schardinger, F.Untersuch Nahrungs Genussmittel,,. Beinert, H.Biochem. Soc. Trans.,,. Bray, R.C.Q. Rev. Biophys.,,. Massey, V. & Harris, C.M.Biochem. Soc. Trans.,,. Elion, G.B.Science,,. Hille, R. & Nishino, T.Faseb J.,,. Nishino, T.J. Biochem. Tokyo,,. McCord, J.M.N.Engl.J.Med.,,. Vorbach, C., Scriven, A., & Capecchi, M.R.Genes Dev.,,. Enroth, C., Eger, B.T., Okamoto, K., Nishino, T., Nishino, T., &Pai,E.F.Proc. Natl. Acad. Sci. USA,,. Kuwabara, Y., Nishino, T., Okamoto, K., Matsumura, T., Eger, B.T., Pai, E.F., & Nishino, T.Proc. Natl. Acad. Sci. USA,,. Nishino, T., Okamoto, K., Kawaguchi, Y., Hori, H., Matsumura, T., Eger, B.T., Pai, E.F., & Nishino, T.J. Biol. Chem.,,. Asai, R., Nishino, T., Matsumura, T., Okamoto, K., Igarashi, K., Pai, E.F., & Nishino, T.J. Biochem. Tokyo,,. Nishino, T.FEBS J., in press. Nishino, T., Amaya, Y., Kawamoto, S., Kashima, Y., Okamoto, K., & Nishino, T.J. Biochem. Tokyo,,. Hille, R., Hagen, W.R., & Dunham, W.R.J. Biol. Chem.,,. Palmer,G.&Massey,V.J. Biol. Chem.,,. Bray, R.C.in The Enzymes Boyer, P.D. ed., vol., pp., Academic Press, New York. Truglio, J.J., Theis, K., Leimkuhler, S., Rappa, R., Rajagopalan, K.V., & Kisker, C.Structure,,. Cramer, S.P., Johnson, J.L., Rajagopalan, K.V., & Sorrell, T.N. Biochem. Biophys. Res. Commun.,,. Romao, M.J., Archer, M., Moura, I., Moura, J.J., LeGall, J., Engh, R., Schneider, M., Hof, P., & Huber, R.Science,

9 ,. Huber, R., Hof, P., Duarte, R.O., Moura, J.J., Moura, I., Liu, M.Y., LeGall, J., Hille, R., Archer, M., & Romao, M.J. Proc. Natl. Acad. Sci. USA,,. Okamoto, K., Matsumoto, K., Hille, R., Eger, B.T., Pai, E.F., & Nishino, T.Proc. Natl. Acad. Sci. USA,,. Jones, R.M., Inscore, F.E., Hille, R., & Kirk, M.L.Inorg Chem.,,. Doonan, C.J., Stockert, A., Hille, R., & George, G.N. J. Am. Chem. Soc.,,. Holm, R.H.Cood. Chem. Rev.,,. Avis, P.G., Bergel, F., Bray, R.C., & Shooter, K.V. Nature,,. Nishino, T., Nishino, T., & Tsushima, K.FEBS Lett.,,. Eger, B.T., Okamoto, K., Enroth, C., Sato, M., Nishino, T., Pai, E.F., & Nishino, T.Acta Crystallogr. D Biol. Crystallogr.,,. Iwasaki, T., Okamoto, K., Nishino, T., Mizushima, J., & Hori, H.J. Biochem. Tokyo,,. Nishino, T. & Okamoto, K.J. Inorg. Biochem.,,. Okamoto, K., Eger, B.T., Nishino, T., Kondo, S., Pai, E.F., & Nishino, T.J. Biol. Chem.,,. Fukunari, A., Okamoto, K., Nishino, T., Eger, B.T., Pai, E.F., Kamezawa, M., Yamada, I., & Kato, N.J. Pharmacol. Exp. Ther.,,. Okamoto, K., Eger, B.T., Nishino, T., Pai, E.F., & Nishino, T. Nucleosides, Nucleotides and Nucleic Acids, in press. Leimkuhler, S., Stockert, A.L., Igarashi, K., Nishino, T., & Hille, R.J. Biol. Chem.,,. Yamaguchi, Y., Matsumura, T., Ichida, K., Okamoto, K., & Nishino, T.J. Biochem. Tokyo,,. Glatigny, A., Hof, P., Romao, M.J., Huber, R., & Scazzocchio, C.J. Mol. Biol.,,. Pauff, J.M., Hemann, C.F., Junemann, N., Leimkuhler, S., & Hille, R.J. Biol. Chem.,,. Xia, M., Dempski, R., & Hille, R.J. Biol. Chem.,,. Maiti, N.C., Tomita, T., Kitagawa, T., Okamoto, K., & Nishino, T.J. Biol. Inorg. Chem.,,. Howes, B.D., Bray, R.C., Richards, R.L., Turner, N.A., Bennett,B.,&Lowe,D.J. Biochemistry,,. Lowe, D.J., Richards, R.L., & Bray, R.C.Biochem. Soc. Trans.,,. Xia, M., Ilich, P., Dempski, R., & Hille, R.Biochem. Soc. Trans.,,. Amano, T., Ochi, N., Sato, H., & Sakaki, S.J. Am. Chem. Soc.,,. Garattini, E., Mendel, R., Romao, M.J., Wright, R., & Terao, M.Biochem. J.,,. Massey, V., Komai, H., Palmer, G., & Elion, G.B.J. Biol. Chem.,,. Huh, K., Yamamoto, I., Gohda, E., & Iwata, H.Jpn. J. Pharmacol.,,. Ichida, K., Kamatani, N., Nishino, T., Saji, M., Okabe, H., & Hosoya, T.Adv. Exp. Med. Biol.,,. Jezewska, M.M.Eur. J. Biochem.,,. Pauff, J.M., Zhang, J., Bell, C.E., & Hille, R.J. Biol. Chem.,,. Lorigan, G.A., Britt, R.D., Kim, J.H., & Hille, R.Biochim. Biophys. Acta,,.

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