Molybdenum and tungsten enzymes: the xanthine oxidase family

Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways.     

Inhibitors of the molybdenum cofactor containing 4-hydroxybenzoyl-CoA reductase

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a member of the xanthine oxidase (XO) family of molybdenum cofactor containing enzymes and catalyzes the irreversible removal of a phenolic hydroxy group by reduction, yielding benzoyl-CoA and water. In this work the effects of various activity modulating compounds were characterized by kinetic, electron paramagnetic resonance (EPR) spectroscopic, and X-ray crystallographic studies. 4-HBCR was readily inactivated by cyanide and by the reducing agents titanium(III) citrate and dithionite; in contrast, reduced viologens had no inhibitory effect. Cyanide inhibition occurred in both the oxidized and reduced state of 4-HBCR. In the reduced state, cyanide-inhibited 4-HBCR was reactivated by simple oxidation. In contrast, reactivation from the oxidized state was only achieved in the presence of sulfide. Dithionite-inhibited 4-HBCR was reactivated by oxidation, whereas inhibition by titanium(III) citrate was irreversible. The previously reported inhibitory effect of azide could not be confirmed; instead, azide rather protected the enzyme from inactivation by titanium(III) citrate. The EPR spectra of the Mo(V) states were nearly identical in the noninhibited methyl viologen and in the dithionite-inhibited states of 4-HBCR; they exhibited a hyperfine splitting due to magnetic coupling with two solvent-exchangeable protons. The cyanide-treated enzyme showed the typical desulfo-inhibited Mo(V) EPR signal in D 2O, whereas in H 2O the hyperfine splitting was altered but indicated no loss of Mo(V)-proton interactions. The structures of dithionite- and azide-bound 4-HBCR were solved at 2.1 and 2.2 A, respectively. Both dithionite and azide bound directly to equatorial ligation sites of the Mo atom. The results obtained revealed further insights into the active site of an unusual member of the XO family of molybdenum cofactor containing enzymes.     

Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: mechanistic implications.

For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.     

Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow

The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis.     

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.     

Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis

The lpdA (Rv3303c) gene from Mycobacterium tuberculosis encoding a new member of the flavoprotein disulfide reductases was expressed in Escherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+ per monomer. Although annotated as a probable lipoamide dehydrogenase in M. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2H]NADH establishing that the enzyme promotes transfer of the C4-proS hydride of NADH. The absence of an isotope effect with [4S-2H]NADPH, the low Km value of 0.5 microm for NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+ (Ki approximately 6 nm) and 2'-phospho-ADP-ribose (Ki approximately 800 nm), demonstrate the high affinity of LpdA for 2'-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+ rather than NADH/NAD+. Modeling of NADP+ in the active site revealed that LpdA achieves the high specificity for NADP+ through interactions involving the 2'-phosphate of NADP+ and amino acid residues that are different from those in glutathione reductase.     

Spectroscopic studies on the iron-sulfur centers of milk xanthine oxidase

The optical electron paramagnetic resonance and M?ssbauer spectral properties of the two iron-sulfur centers present in milk xanthine oxidase have been reexamined. It is found in the case of the optical spectral change observed on reduction of the enzyme that the two centers contribute approximately equally, with a ratio of spectral contributions for Fe/S I and Fe/S II of 0.55:0.45. This conclusion is based both on the behavior of the spectral change at wavelengths where only the two iron-sulfur centers contribute to the spectral change (under experimental conditions minimizing the effect of flavin semiquinone) during reductive titrations and a comparison of the spectra of 1- and 2-electron reduced enzyme under different conditions. This very similar spectral weighting for the two centers applies throughout the visible region. In the case of the EPR spectra, it is found from computer simulation of the signals observed under nonsaturating conditions that iron-sulfur center II exhibits g values of 1.902, 1.991, and 2.110 and does not exhibit two g values above that for the free electron, as has been reported (Lowe, J., Lynden-Bell, R.M., and Bray, R. C. (1972) Biochem. J. 130, 239-249). The g values for iron-sulfur center I obtained from the simulations are 1.894, 1.932, and 2.022. Finally, M?ssbauer spectra of xanthine oxidase have been obtained, and it is found that while the two iron-sulfur centers are indistinguishable in the oxidized state, the ferrous iron in one of the reduced iron-sulfur centers exhibits an unusually large quadrupole coupling.     

3,5,2',4'-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver–Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.     

Pyranose oxidase identified as a member of the GMC oxidoreductase family

Fungal pyranose oxidase is a flavoenzyme whose preferred substrate among several monosaccharides is D-glucose. After a comprehensive analysis of conserved features in a structure-based multiple sequence alignment of homologous proteins, we could classify this enzyme into the GMC oxidoreductase family. The identified homology also suggests a three-dimensional protein structure similar to the functionally related glucose oxidase.     

Functional groups involved in the nitrate reductase activity of milk xanthine oxidase

Milk xanthine oxidase possesses the nitrate reductase activity at pH 5.2; the pH optimum of the xanthine oxidase activity for the enzyme lies at 9.6. After removal of FAD and binding of Mo and Fe with a simultaneous measurement at the pH optima of the above activities it was found that only the Mo-containing site is necessary for the nitrate reductase activity. The switch-over of the enzyme from the xanthine oxidase to the nitrate reductase activity is associated with considerable conformational changes of the enzyme molecule.     

Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.     

Resonance Raman studies of the flavin and iron-sulfur centers of milk xanthine oxidase

Resonance Raman spectroscopy has been used to study milk xanthine oxidase, an enzyme containing molybdenum, binuclear iron-sulfur clusters, and FAD as cofactors. The contribution of FAD dominates the resonance Raman spectrum at frequencies above 500 cm-1. As expected, no bands assignable to FAD are observed in deflavo xanthine oxidase. The resonance Raman spectrum below 500 cm-1 reveals the contribution of the Fe2S2(Cys)4 groups with frequencies similar to those of adrenodoxin and putidaredoxin. Resonance enhancement profiles of the Fe2S2(Cys)4 clusters indicate intensity variations among the Fe2S2(Cys)4 peaks that are attributed to different excitation wavelength maxima of their bridging and terminal iron-sulfur vibrations. No evidence for Mo-ligand vibrations could be obtained by using excitation wavelengths between 363.8 and 514.5 nm.     

Amino acid sequence of long chain alpha-hydroxy acid oxidase from rat kidney, a member of the family of FMN-dependent alpha-hydroxy acid-oxidizing enzymes

The complete amino acid sequence of rat kidney long chain alpha-hydroxy acid oxidase has been determined by microsequencing, using a number of standard enzymatic and chemical cleavages. Peptides were purified by high pressure liquid chromatography or by gel electrophoresis followed by electrotransfer. The sequence comprises 352 residues and ends with a peroxisomal targeting sequence SRL. The present work definitely establishes that hydroxy acid oxidase is a member of the family of FMN-dependent alpha-hydroxy acid-oxidizing enzymes. The family includes lactate oxidase, short chain alpha-hydroxy acid oxidase (glycolate oxidase), flavocytochrome b2, and mandelate dehydrogenase. There are altogether 45 totally conserved positions among the six sequences known. The sequence similarities are analyzed in light of the known three-dimensional structure of flavocytochrome b2 and glycolate oxidase. It is concluded that long chain hydroxy acid oxidase should be folded as a beta 8 alpha 8 barrel and should dehydrogenate alpha-hydroxy acids according to the same chemical mechanism as other enzymes of the family, in spite of a Tyr----Phe substitution at the active site.     

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues

Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.     

On the mechanism of action of xanthine oxidase. Evidence in support of an oxo transfer mechanism in the molybdenum-containing hydroxylases

The mechanism of action of xanthine oxidase has been investigated using single-turnover experiments in an effort to determine the primary source of the oxygen atom incorporated into product in the course of catalysis. It is found from mass spectroscopic analysis of the uric acid generated in these experiments that when 16O-labeled enzyme in [18O]H2O is reacted with substoichiometric amounts of xanthine (under conditions where no enzyme molecule is likely to react with more than one substrate molecule), the uric acid isolated from the reaction mixture contains 16O at position 8 of the purine ring. Conversely, when 18O-labeled enzyme in [16O]H2O is exposed to substoichiometric xanthine, 18O is incorporated into the product uric acid. These results strongly support a variety of chemical studies with model molybdenum complexes suggesting that the oxygen atom of the Mo = O group known to be present at the active site of xanthine oxidase is transferred to product in the course of catalysis. The mechanistic implications of the present work are discussed.