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Properties of xanthine dehydrogenase that lacks iron-sulfur centers
Journal of Inorganic Biochemistry
J18
IRON-SULFUR AND O T H E R METAL-SULFUR CLUSTERS
PROPERTIES OF XANTHINE DEHYDROGENASE LACKS IRON-SULFUR CENTERS
263
THAT
K. Okamoto a, T. Iwasaki a, T. Nishino b, H. Hori% J. Mizushima ~ and T. Nishino a
aDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi 115, BunkyoKu, Tokyo 113, Japan; bDepartment of Biochemistry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-Ku, Yokohama 236, Japan Xanthine dehydrogenase is a homodimer of molecular weight 300,000 and each subunit has one FAD, one molybdopterin and two [2Fe-2S] centers. The enzyme oxidizes xanthine to uric acid at the molybdenum with subsequent reduction of NAD to NADH at FAD, therefore the intramolecular electron transfer is indispensable for such catalytic cycle, but the role of two iron-sulfur centers still remains to be obscure. The visible absorption spectra of these two iron-sulfur centers are indistinguishable, but their EPR properties are quite different. One of the iron-sulfur centers designated Fe/S II has approximately 80 mV higher redox potential than that of the other iron-suffer center, and its EPR signal is unusually broad and can be observed only below 25 K. The other iron-sulfur center designated Fe/S I elicits a rhombic EPR signal that is very similar to those of the [2Fe-2S] type ferredoxins. Because the redox potentials of two iron-sulfur centers are largely different (Fe/S I = -310mV, Fe/S II = -236mV) [1], it has been suggested that the functions of these centers during enzyme are different. Pulse radiolysis analysis of milk xanthine dehydrogenase showed that an electron transfers from molybdenum to FAD directly and that rapid electron equilibrium between flavin semiquinone and one of the iron-sulfur centers occurs, suggesting that this iron-sulfur center modulates the reactivity of FAD with oxygen [2]. We constructed some mutant enzymes of rat liver xanthine dehydrogenase (C51S, C115S and C115A) and expressed them in insect cells. These two mutated residues are deduced to be the ligands of the two iron-sulfur centers from amino acid sequence comparison of xanthine dehydrogenase of different species and from X-ray crystal structure analysis of xanthine dehydrogenase related enzyme, aldehyde oxidoreductase from Desulfovibrio gigas [3]. C l l 5 A mutant had xanthine-NAD activity which is compatible with the speculation that one of the iron-sulfur centers is in the reduced state during the turnover reaction of the enzyme and does not have any role in the catalytic reaction [2, 4]. The expressed enzymes consist of heterogeneous species of enzyme molecules. Only few percent of the expressed enzyme was active form, i.e. native form, and the rest was largely obtained as de-molybdo form, lacking molybdopterin. Approximately a half of the de-molybdo enzyme was dimeric form, and the other half was monomeric form that has not been available naturally. The monomeric forms of the wild type enzyme and the Cys-115 mutated enzymes are suggested to contain only one [2Fe-2S] center per molecule as judged by the atomic absorption and EPR spectra. They elicited a broad and rhombic EPR signal which is attributable to a [2Fe-2S] cluster but is distinctively different from that of either Fe/S I or Fe/S II of the native, dimeric enzyme. The EPR spectra of the dimeric forms of C51S and C 115S mutants showed that they contain two distinctive [2Fe-2S] centers, suggesting the serine ligand coordination to one of the iron-sulfur centers. The principle g values of both centers of C51S were indistinguishable from those of native enzyme, although those of Fe/S I center of C115S were markedly different. 1. J. Hunt, V. Massey, W. R. Dunham and R. H. Sands, J. BioL Chem. 268, 18685 (1993) 2. K. Kobayashi, M. Miki, K. Okamoto and T. Nishino, J. Biol. Chem. 268, 24642 (1993) 3. M. J. Romao, M. Archer, I. Moura, J. J. G. Moura, J. LeGall, R. Engh, M. Schneider, P. Hof and R. Huber, Science. 270, 1170 (1995) 4. T. Nishino, T. Nishino, L. M. Schopfer and V. Massey, J. Biol. Chem. 264, 2518 (1989)
J18
IRON-SULFUR AND O T H E R METAL-SULFUR CLUSTERS
PROPERTIES OF XANTHINE DEHYDROGENASE LACKS IRON-SULFUR CENTERS
263
THAT
K. Okamoto a, T. Iwasaki a, T. Nishino b, H. Hori% J. Mizushima ~ and T. Nishino a
aDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi 115, BunkyoKu, Tokyo 113, Japan; bDepartment of Biochemistry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-Ku, Yokohama 236, Japan Xanthine dehydrogenase is a homodimer of molecular weight 300,000 and each subunit has one FAD, one molybdopterin and two [2Fe-2S] centers. The enzyme oxidizes xanthine to uric acid at the molybdenum with subsequent reduction of NAD to NADH at FAD, therefore the intramolecular electron transfer is indispensable for such catalytic cycle, but the role of two iron-sulfur centers still remains to be obscure. The visible absorption spectra of these two iron-sulfur centers are indistinguishable, but their EPR properties are quite different. One of the iron-sulfur centers designated Fe/S II has approximately 80 mV higher redox potential than that of the other iron-suffer center, and its EPR signal is unusually broad and can be observed only below 25 K. The other iron-sulfur center designated Fe/S I elicits a rhombic EPR signal that is very similar to those of the [2Fe-2S] type ferredoxins. Because the redox potentials of two iron-sulfur centers are largely different (Fe/S I = -310mV, Fe/S II = -236mV) [1], it has been suggested that the functions of these centers during enzyme are different. Pulse radiolysis analysis of milk xanthine dehydrogenase showed that an electron transfers from molybdenum to FAD directly and that rapid electron equilibrium between flavin semiquinone and one of the iron-sulfur centers occurs, suggesting that this iron-sulfur center modulates the reactivity of FAD with oxygen [2]. We constructed some mutant enzymes of rat liver xanthine dehydrogenase (C51S, C115S and C115A) and expressed them in insect cells. These two mutated residues are deduced to be the ligands of the two iron-sulfur centers from amino acid sequence comparison of xanthine dehydrogenase of different species and from X-ray crystal structure analysis of xanthine dehydrogenase related enzyme, aldehyde oxidoreductase from Desulfovibrio gigas [3]. C l l 5 A mutant had xanthine-NAD activity which is compatible with the speculation that one of the iron-sulfur centers is in the reduced state during the turnover reaction of the enzyme and does not have any role in the catalytic reaction [2, 4]. The expressed enzymes consist of heterogeneous species of enzyme molecules. Only few percent of the expressed enzyme was active form, i.e. native form, and the rest was largely obtained as de-molybdo form, lacking molybdopterin. Approximately a half of the de-molybdo enzyme was dimeric form, and the other half was monomeric form that has not been available naturally. The monomeric forms of the wild type enzyme and the Cys-115 mutated enzymes are suggested to contain only one [2Fe-2S] center per molecule as judged by the atomic absorption and EPR spectra. They elicited a broad and rhombic EPR signal which is attributable to a [2Fe-2S] cluster but is distinctively different from that of either Fe/S I or Fe/S II of the native, dimeric enzyme. The EPR spectra of the dimeric forms of C51S and C 115S mutants showed that they contain two distinctive [2Fe-2S] centers, suggesting the serine ligand coordination to one of the iron-sulfur centers. The principle g values of both centers of C51S were indistinguishable from those of native enzyme, although those of Fe/S I center of C115S were markedly different. 1. J. Hunt, V. Massey, W. R. Dunham and R. H. Sands, J. BioL Chem. 268, 18685 (1993) 2. K. Kobayashi, M. Miki, K. Okamoto and T. Nishino, J. Biol. Chem. 268, 24642 (1993) 3. M. J. Romao, M. Archer, I. Moura, J. J. G. Moura, J. LeGall, R. Engh, M. Schneider, P. Hof and R. Huber, Science. 270, 1170 (1995) 4. T. Nishino, T. Nishino, L. M. Schopfer and V. Massey, J. Biol. Chem. 264, 2518 (1989)