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

Abstract

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) Science 270, 1170-1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals: the less anisotropic one, called signal I, is generally similar to the g(av) approximately 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.

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

“…Magnetic couplings between two paramagnetic centers showing different relaxation times give temperature-dependent splitting of the resonance lines of the center relaxing slower. [48][49][50] For the case of the slow-type signal, the nearly isotropic splitting is completely averaged out at temperatures ∼100 K or above [FeS1 relaxes faster than Mo(V)]. [21][22][23] This is not the case of the alcohol-inhibited DgAOR signals (Figure 2), which demonstrates that the larger line width of the "red" signals with respect to the "reox" signals at 140 K is due to the larger magnetic coupling between Mo(V) and FeS1 centers.…”

Structural and Electron Paramagnetic Resonance (EPR) Studies of Mononuclear Molybdenum Enzymes from Sulfate-Reducing Bacteria

“…Similar ideas were used to assign the proximal FeS center in the proteins of the XO family Dg Aor and BXDH but with a model based on the semiempirical Bloch equations. 37 …”

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

“…FeS I, the proximal center, is closer to the Mo site and is buried inside the protein, whereas FeS II, the distal center, is situated near the surface of the protein. Both iron-sulfur centers and a fraction of the molybdenum ions are paramagnetic in the reduced state of the protein and, in addition, show intercenter magnetic couplings [25][26][27]. The currently accepted mechanism implies substrate interaction with the Mo center, which is then reduced from Mo(VI) to Mo(IV), followed by a two electron transfer to an external electron acceptor, in a process mediated by the two iron-sulfur centers.…”