Electronic structure contributions to reactivity in xanthine oxidase family enzymes.

We review the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes with a specific emphasis on electronic structure contributions to reactivity. In addition to xanthine and aldehyde oxidoreductases, which catalyze the two-electron oxidation of aromatic heterocycles and aldehyde substrates, this mini-review highlights recent work on the closely related carbon monoxide dehydrogenase (CODH) that catalyzes the oxidation of CO using a unique Mo-Cu heterobimetallic active site. A primary focus of this mini-review relates to how spectroscopy and computational methods have been used to develop an understanding of critical relationships between geometric structure, electronic structure, and catalytic function.

Variation of average g values and effective exchange coupling constants among [2Fe-2S] clusters: a density functional theory study of the impact of localization (trapping forces) versus delocalization (double-exchange) as competing factors.

A phenomenological model aimed at rationalizing variations in both average g-tensor values (gav identical with 1/3Sigmaigi ) and effective exchange coupling constants Jeff (defined as two-thirds of the energy difference between the S = 3/2 and S = 1/2 spin states) has been derived in order to describe the great variety of magnetic properties exhibited by reduced [2Fe-2S] clusters in proteins. The key quantity in the present analysis is the ratio Delta E/B computed from two competing terms. Delta Ecomprises various effects that result in trapping-site asymmetries: vibronic coupling and the chemical nature (S/N/O) and conformations of the ligands on the one hand and solvation terms, the hydrogen bonding network, etc., on the other. All of these additive terms (in a "bottom-up" approach) favor valence localization of the reducing electron onto one of the two iron sites. In contrast, the B term is the double-exchange term, which favors electronic delocalization. Both gav and Jeff can be expressed as functions of Delta E/ B. We have also shown that electronic localization generally favors small gav and large Jeff values (while the opposite is true for electronic delocalization) in a comparative study of the spectroscopic features of plant-type ferredoxins (Fd's) and Rieske centers (and related mutants). Two other types of problems were particularly challenging. The first of these involved deprotonated Rieske centers and the xanthine oxidase clusters II, which are characterized by very small Jeff values (40-45 cm (-1) with a J S A. S B model) correlated with unusually large gav values (in the range 1.97-2.01) as a result of an antisymmetric exchange coupling mechanism. The second concerned the analogous Fd's from Clostridium pasteurianum (Cp) and Aquifex aeolicus (Aa). Detailed Mössbauer studies of the C56S mutant of the Cp system revealed a mixture of clusters with valence-localized S = 1/2 and valence-delocalized S = 9/2 ground states. We relied on crystallographic structures of wild-type and mutant Aa Fd's in order to explain such a distribution of spin states.

Direct electron transfer of xanthine oxidase and its catalytic reduction to nitrate.

This work reports on the direct electrochemistry of the xanthine oxidase (XO) from buttermilk, a mononuclear molybdenum enzyme that comprises four redox active cofactors: a five-coordinate mononuclear Mo ion, two [2Fe-2S] clusters, and a flavin adenine dinucleotide (FAD) group. The Mo, [2Fe-2S] and FAD redox responses are obtained from the enzyme immobilized on an activated single-wall carbon nanotubes (SWNTs) modified glassy carbon electrode using protein film voltammetry. The formal potentials of which are -0.61 V, -0.47 V and -0.37 V (vs. SCE) at pH 5.0, respectively. Upon addition of nitrate to the electrochemical cell, a steady-state voltammogram and i-t amprometric response were observed, indicating XO can catalyze the reduction of nitrate.

A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid.

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.