Using a myosin preparation containing endogenous myosin light-chain (LC2) kinase and phosphatase and calmodulin, i.e. near physiological ones, the interaction of vanadate oligomers with phosphorylated myosin was evaluated. Decavanadate or metavanadate solutions (2-15 mM total vanadate) did not prevent the phosphorylation state of the regulatory myosin lightchain, as observed by urea-polyacrylamide gel electrophoresis. The relative order of line broadening upon protein addition, reflecting the interaction of the vanadate oligomers with phosphorylated myosin, was V10 > V-4 > V-1 = 1 whereas, no changes were observed for monomeric vanadate. In the presence of ATP, V-1 signal was shifted upfield 2 ppm and became broadened, while V4 signal became narrowed. Moreover, a significant increase in myosin ATPase inhibition (60%) was observed when decameric vanadate species were present (1.4 mM). It is concluded that, under conditions near physiological ones, decameric vanadate differs from vanadate oligomers present in metavanadate solutions due to its strong interaction with the phosphorylated enzyme and myosin ATPase inhibition. Besides, ATP decreases the affinity of myosin for tetravanadate, induces the interaction with monomeric vanadate, whereas it does not affect decameric vanadate interaction. (C) 2002 Elsevier Science B.V. All rights reserved.

Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.

The tungsten-containing formate dehydrogenase (W-FDH) isolated from Desulfovibrio gigas has been crystallized in space group P2(1), with cell parameters a = 73.8 A, b = 111.3 A, c = 156.6 A and beta = 93.7 degrees. These crystals diffract to beyond 2.0 A on a synchrotron radiation source. W-FDH is a heterodimer (92 kDa and 29 kDa subunits) and two W-FDH molecules are present in the asymmetric unit. Although a molecular replacement solution was found using the periplasmic nitrate reductase as a search model, additional phasing information was needed. A multiple-wavelength anomalous dispersion (MAD) dataset was collected at the W- and Fe-edges, at four different wavelengths. Anomalous and dispersive difference data allowed us to unambiguously identify the metal atoms bound to W-FDH as one W atom with a Se-cysteine ligand as well as one [4Fe-4S] cluster in the 92 kDa subunit, and three additional [4Fe-4S] centers in the smaller 29 kDa subunit. The D. gigas W-FDH was previously characterized based on metal analysis and spectroscopic data. One W atom was predicted to be bound to two molybdopterin guanine dinucleotide (MGD) pterin cofactors and two [4Fe-4S] centers were proposed to be present. The crystallographic data now reported reveal a selenium atom (as a Se-cysteine) coordinating to the W site, as well as two extra [4Fe-4S] clusters not anticipated before. The EPR data were re-evaluated in the light of these new results.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate specificity similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6(1)22, with unit cell dimensions of a=b=156.4 A and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

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.

We report on a new method to make nanostructures in aqueous solution at room temperature. We used the protein cytochrome c(3) to catalyze reduction of selenate (SeO42-) to selenium Se-0 by dithionite. Reduction was instantaneous. After a week spherical nanoparticles of red Se-0 (about 50 nm diameter) precipitated, followed by self-assembling into crystalline nanowires, typically 1 mu m long. The nanowires were composed of one strand of spherical particles; thicker strands contained several nanoparticles in parallel.

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Romao, M. J., Knablein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10(-8) - 10(-2) M range with a detection limit of 10(-8) M. and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NIR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10(-5) - 10(-2) M) and a higher detection limit (10(-5) M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)(4) metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A. Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55-60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) = 32 microM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3-C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD).

The regulation of the properties of Desulfovibrio gigas flavodoxin in AOT/water/iso-octane micellar system was studied. UV-visible spectroscopic studies have shown that photoreduction of flavodoxin in the presence of EDTA leads to hydroquinone formation through the intermediate semiquinone. The [free FMN] - [bound to flavodoxin FMN] equilibrium (and hence, the amount of apoprotein) depends on redox state of FMN and on hydration degree which controls the micellar size. Thus, a new method of reversible cofactor removing under mild conditions (at low hydration degree of micelles) is suggested, accompained by isolation of apo-form of the protein.

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD(+)-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580-587) is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. The nickel K-edge energies shift 0.9-1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (-0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent intermediate redox level (SI). Analysis of the XANES features assigned to 1s-->3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The smallest edge energy shift is observed for the T. roseopersicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample of A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at similar to 2.2 Angstrom. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3-4 N,O-donor atoms at 2.06(2) Angstrom, with contributions from 2-3 S-donor ligands at 2.35(2) Angstrom. This changes upon reduction to a more typical nickel site composed of similar to 4 S-donor ligands at a Ni-S distance of 2.19(2) Angstrom. Evidence for the presence of atoms in the 2.4-2.9 Angstrom distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to distinguish between S and Fe scattering atoms at this distance, and by the potential presence of both S and another metal atom at similar distances. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of the D. gigas enzyme.

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.