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

Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S](1+,2+) c lusters 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 su rface, 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., L eGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 2 70, 1170-1176]. These two clusters display distinct electron paramagnetic r esonance (EPR) signals: the less anisotropic one, called signal I, is gener ally similar to the g(av) approximate to 1.96-type signals given by ferredo xins, whereas signal II often exhibits anomalous properties such as very la rge g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin-lattice relaxation tim e and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR prope rties of signal II arise from the presence of low-lying excited levels refl ecting significant double exchange interactions. The issue raised by the as signment 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 depen dence of this signal could be quantitatively reproduced by assuming that th e Mo(V) center is coupled to the cluster giving signal I in xanthine oxidas e as well as in D, gigas aldehyde oxidoreductase. This demonstrates unambig uously that, in both enzymes, signal I arises from the center which is clos est to the molybdenun cofactor.