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Analysis of the Electron Paramagnetic Resonance Properties of the [2Fe-2S]1+ Centers in Molybdenum Enzymes of the Xanthine Oxidase Family:  Assignment of Signals I and II

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Instituto Superior de Ciências da Saùde-Sul and Departamento de Quimica, Centro de Quimica Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Caparica, Portugal, Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, and Université de Provence, 13331 Marseille Cedex 3, France
Cite this: Biochemistry 2000, 39, 10, 2700–2707
Publication Date (Web):February 15, 2000
Copyright © 2000 American Chemical Society

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    Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S]1+,2+ clusters that are bound to the protein by very different cysteine motifs. In the X-ray crystal structure of Desulfovibrio gigas aldehyde oxidoreductase, the cluster ligated by a ferredoxin-type motif is close to the protein surface, whereas that ligated by an unusual cysteine motif is in contact with the molybdopterin [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science270, 1170−1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals:  the less anisotropic one, called signal I, is generally similar to the gav ≈ 1.96-type signals given by ferredoxins, whereas signal II often exhibits anomalous properties such as very large g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin−lattice relaxation time and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR properties of signal II arise from the presence of low-lying excited levels reflecting significant double exchange interactions. The issue raised by the assignment of signals I and II to the two [2Fe-2S]1+ clusters was solved by using the EPR signal of the Mo(V) center as a probe. The temperature dependence of this signal could be quantitatively reproduced by assuming that the Mo(V) center is coupled to the cluster giving signal I in xanthine oxidase as well as in D. gigas aldehyde oxidoreductase. This demonstrates unambiguously that, in both enzymes, signal I arises from the center which is closest to the molybdenum cofactor.

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     Instituto Superior de Ciências da Saùde-Sul.


     Universidade Nova de Lisboa.

     Institut de Biologie Structurale et Microbiologie, CNRS, and Université de Provence.


     Corresponding author:  Fax (33) 4 91 16 45 78; E-mail [email protected].

     This work was supported in part by the ICCTI/CNRS exchange program.

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