The inhibition of xanthine oxidase by 8-bromoxanthine.

The interaction of xanthine oxidase with the substrate analog 8-bromoxanthine has been examined in an effort to determine the nature of interaction of purines with the active site of the enzyme. It is found that 8-bromoxanthine is an inhibitor of xanthine oxidase with a Ki of approximately 400 microM; inhibition is uncompetitive with respect to xanthine and noncompetitive with respect to molecular oxygen. While 8-bromoxanthine has only a slight effect on the reaction of reduced enzyme with oxygen, it dramatically slows the rate of enzyme reduction by xanthine, suggesting that inhibition does involve the interaction of 8-bromoxanthine with the molybdenum center of the enzyme. KD determinations for binding of 8-bromoxanthine to oxidized and reduced xanthine oxidase indicate that the inhibitor binds preferentially to the fully reduced form of the molybdenum center (MoIV), with dissociation constants of 1.5 mM and 18 microM for oxidized and reduced enzyme, respectively. This preferential binding to the reduced form of the enzyme is manifested in a significant increase in the oxidation-reduction potentials of the molybdenum center as determined by potentiometric titrations with 8-bromoxanthine complexed with xanthine oxidase. The shape of the Mov EPR signal observed in the course of these titrations as well as a comparison with results of reductive titrations and KD determinations with uric acid and xanthine indicate that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase in a way that is typical of purine substrates and products, despite the presence of the bulky Br group. The inhibitor thus has a potential as a probe of enzyme-substrate interactions, particularly using the technique of x-ray absorption spectroscopy.

The Inhibition of Xanthine Oxidase by 8=Bromoxanthine* (Received for publication, August 24, 1983) Russ HilleS and Richard C. Stewart4 From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109 The interaction of xanthine oxidase with the substrate analog 8-bromoxanthine has been examined in an effort to determine the nature of interaction of purines with the active site of the enzyme. It is found that 8-bromoxanthine is an inhibitor of xanthine oxidase with a Ki of approximately 400 p~; inhibition is uncompetitive with respect to xanthine and noncompetitive with respect to molecular oxygen. While 8bromoxanthine has only a slight effect on the reaction of reduced enzyme with oxygen, it dramatically slows the rate of enzyme reduction by xanthine, suggesting that inhibition does involve the interaction of 8-bromoxanthine with the molybdenum center of the enzyme. K D determinations for binding of 8-bromoxanthine to oxidized and reduced xanthine oxidase indicate that the inhibitor binds preferentially to the fully reduced form of the molybdenum center (Mo'"), with dissociation constants of 1.5 mM and 18 PM for oxidized and reduced enzyme, respectively. This preferential binding to the reduced form of the enzyme is manifested in a significant increase in the oxidation-reduction potentials of the molybdenum center as determined by potentiometric titrations with 8-bromoxanthine complexed with xanthine oxidase. The shape of the Mo" EPR signal observed in the course of these titrations as well as a comparison with results of reductive titrations and KO determinations with uric acid and xanthine indicate that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase in a way that is typical of purine substrates and products, despite the presence of the bulky Br group. The inhibitor thus has a potential as a probe of enzyme-substrate interactions, particularly using the technique of x-ray absorption spectroscopy.
Xanthine oxidase is a complex molybdoflavoprotein that catalyzes the hydroxylation of xanthine to form uric acid. This reaction takes place at the molybdenum center of the enzyme and formally involves the removal of a hydride ion from position 8 of the purine ring followed by its replacement with hydroxide from water. A great deal of research effort has focused on the molybdenum center of xanthine oxidase, primarily using electron paramagnetic resonance spectroscopy (for a comprehensive review, see Ref. 1) but also more recently x-ray absorption spectroscopy (2-4). The precise manner in which substrate interacts with the molybdenum center of the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient (with Dr. Vincent Massey) of National Science Foundation Grant PCM-8208240 in support of this work.
$ Recipient of a National Science Foundation predoctoral fellowship. enzyme and the sequence of events that result in conversion of substrate to product remain only poorly understood, however, and a number of alternative mechanisms have been proposed (5)(6)(7)(8). In an effort to gain further insight into the nature of the molybdenum center of xanthine oxidase, the interaction of enzyme with the substrate analog 8-bromoxanthine has been investigated. This purine derivative, containing a bromine atom at the position normally hydroxylated by enzyme, is found to be an effective inhibitor of xanthine oxidase. By several criteria, however, it is found to interact with enzyme in substantiaIIy the same manner as xanthine and uric acid and is thus potentially of use in establishing the nature of the enzyme-substrate interaction at the molybdenum center.

MATERIALS AND METHODS
Xanthine oxidase was isolated from fresh unpasteurized milk by the method of Massey et al. (9) with the addition of the folate affinity chromatography procedure described by Nishino et al. (10) as a final step. Enzyme thus obtained was approximately 70% active, having an AFR value in the range of 140-150. (AFR is defined as the ratio of the absorbance change per min at 295 nm monitoring the conversion of xanthine to uric acid divided by the enzyme absorbance at 450 nm in the assay mix. Under the conditions of the standard assay, 111 N M xanthine, 200 @ t M oxygen, 25 "c, in 0.1 M pyrophosphate, 0.3 mM EDTA, pH 8.5, fully active enzyme has an AFR of 210; see ref. 11.) When necessary, active xanthine oxidase was separated from the inactive form of the enzyme as described by Nishino et al. (10). Using this procedure, enzyme exhibiting an AFR in excess of 200 (i.e. was greater than 95% active) could routinely be obtained.
8-Bromoxanthine was prepared by refluxing 1 g of xanthine in 4 g of bromine in a sealed glass tube for 24 h at 90 "C as described by Fischer and Reese (12). After recrystallization from dilute NH40H a white powder was obtained that exhibited the spectral properties of 8-bromoxanthine in both 0.1 N HCl (Amax = 274 nm) and N KOH (Amax = 287 nm). The purity of the reaction product was confirmed by thin layer chromatography using butanol, acetic acid, and water in the ratio 15:3:5 as the developing solvent. In this system, 8-bromoxanthine exhibited an RF of 0.65 while xanthine and uric acid exhibited the same RF value of 0.49.
Optical absorption spectra were recorded with a Cary model 118 or 219 spectrophotometer. Room temperature electron paramagnetic resonance (EPR) spectra were recorded with a Varian 112 X-band spectrometer equipped with a TE 102 cavity. Spectra were recorded at ambient temperature with the sample in a quartz EPR flat cell in order to minimize absorption of the microwave radiation by solvent water. Room temperature potentiometric EPR titrations were performed using an apparatus similar to that described by Porras and Palmer (13) using sodium dithionite as reductant. Operating conditions were: field set 3450 G; scan range, 400 G; time constant, 0.25 s; modulation amplitude, 8 G; modulation frequency, 100 KHz; microwave power, 50 milliwatts; microwave frequency, 9.5 GHz. EPR signals were integrated using the method described by Fee (14) to determine the concentration of paramagnetic species. Spectra at 100 ardized by titration against lumiflavin 3-acetate (16).
Kinetic experiments were performed using a stopped flow spectrophotometer designed and built by Dr. David P. Ballou, Department of Biological Chemistry, The University of Michigan. This instrument was placed on-line to a Data General Nova 2 minicomputer, as described by Entsch et al. (17).
Xanthine (Grade V) was purchased from Sigma and uric acid from Eastman. Unless otherwise stated experiments were performed in 0.1 M sodium pyrophosphate, 0.3 mM EDTA, pH 8.5, at 25 'C.

RESULTS AND DISCUSSION
Steady State Inhibition of Xanthine Oxidase by 8-Bromoxanthine-In order to evaluate the inhibitory effect, if any, of 8-bromoxanthine on the activity of xanthine oxidase, two sets of steady state experiments were performed. In the first, the oxygen concentration was held constant at 600 ~L M while the concentrations of xanthine and 8-bromoxanthine were systematically varied. In the second, xanthine was held constant at 150 p~ and the concentrations of oxygen and 8-bromoxanthine varied. In both experiments, the concentration of the substrate held constant was greater than 10-fold its K, (50 pM for oxygen, 10 pM for xanthine; see Refs. 9 and 18), so that the results reflected to a good approximation the extrapolation to infinite substrate concentration. The results of these experiments are shown in Fig. 1. Somewhat surprisingly, it is found that 8-bromoxanthine, an analog of xanthine, is an uncompetitive rather than competitive inhibitor with respect to reducing substrate; the lines at different concentrations of 8-bromoxanthine in the Lineweaver-Burk plot are parallel (Fig. lA). A plot of the y axis intercept from the primary plot uersus the concentration of 8-bromoxanthine respect to oxygen is to be expected, given that the two interact independently of one another at different sites of the enzyme: the oxygen at the FAD (18) and the 8-bromoxanthine at the molybdenum center (see below). It should be pointed out that the K, values for xanthine and oxygen of 11 and 45 p~, respectively, obtained from the steady state data at zero inhibitor concentration are in good agreement with the accepted literature values reported above.
Xanthine oxidase acts via a ping-pong mechanism, with enzyme alternating between oxidized and reduced forms during turnover (9,19). The parallel lines observed in Lineweaver-Burk plots of l / u uersus l/(xanthine) at a series of oxygen concentrations (or vice versa) are to be expected, however, as xanthine and oxygen interact with enzyme at different sites: the molybdenum center and FAD, respectively. Furthermore, the molybdenum center must be oxidized to Mo"' and the FAD site reduced to FADHz for the reductive and oxidative half-reactions, respectively, to take place. A plausible explanation for the uncompetitive inhibition for 8bromoxanthine with respect to xanthine and its noncompetitive inhibition with respect to oxygen is that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase but binds to the reduced form of the site, not its oxidized form as does xanthine. Precedent exists for this type of inhibition pattern in the inhibition of xanthine oxidase by alloxanthine (another substrate analog, which forms an extremely tight complex with Mo'"; see Ref. In both sets of experiments, the substrate held constant was present at a concentration greater than 10-fold its K, and was, therefore, a reasonable approximation to infinite substrate concentration. The fc, values obtained from secondary plots of the y intercept uersza the inhibitor concentration in A and B (insets) were 420 and 380 p~, respectively, and in reasonable agreement. The K,,, values of 11 and 45 p~ for xanthine and oxygen, respectively (obtained from the lines in the absence of inhibitor) are in good agreement with the literature values of 10 and 50 p~, respectively (30). T.N., turnover number. thine-In order to determine whether 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase as suggested by the steady state results, the reaction of anaerobic oxidized enzyme with xanthine in the presence of 8-bromoxanthine was examined kinetically. The results are shown as a plot of the reciprocal of the observed rate constant uersus the reciprocal of the xanthine concentration a t a series of concentrations for 8-bromoxanthine in Fig. 2. It can be seen that 8-bromoxanthine does in fact markedly decrease the rate of enzyme reduction by xanthine. A plot of they axis intercept from the primary plot uersus the concentration of 8-bromoxanthine ( Fig. 2, inset) gives a Ki in the reductive half-reaction of 395 p~, in good agreement with the Ki of some 400 p~ determined in the steady state work.
In contrast to the reductive half-reaction, the reaction of reduced xanthine oxidase with oxygen is affected to a much lesser extent by the addition of 8-bromoxanthine. The rate of the fast phase of reoxidation (19, 21, 22), corresponding to 75% of tb absorbance increase at 450 nm, and its dependence on oxygen concentration were unaffected by the presence of 1.0 mM 8-bromoxanthine (data not shown). The remaining 25% of the reaction was slower in the presence of 8-bromoxanthine, especially above 250 pM oxygen, and multiphasic. This effect is likely a secondary one due to the higher potential at the molybdenum center in the presence of 8-bromoxanthine (see below), rather than a direct effect of 8-bromoxanthine on the rate at which reduced flavin reacts with 02. The net result of the increase in molybdenum potential would be to remove reducing equivalents from the flavin center, slowing the reaction with oxygen with partially reoxidized enzyme species. Because of the complexity of the slow phase, with effects due to inhibitor dissociation from the molybdenum center, the slow phase was not analyzed further than to point out that the observations were qualitatively consistent with inhibitor binding only at the molybdenum center of the enzyme. Taken together with the kinetic results from the reductive half-reaction and the steady state analysis, it is concluded that 8-bromoxanthine inhibits xanthine oxidase by slowing the rate a t which enzyme in reduced by xanthine, but in a Nay that is noncompetitive with respect to xanthine.
Binding of 8-Bromoxanthine to Oxidized and Reduced Xanthine Oxidase-In an effort to determine more explicitly the manner in which 8-bromoxanthine interacts with xanthine oxidase, titrations of both oxidized and reduced enzyme with the inhibitor were performed (Fig. 3, A and B, respectively).
8-Bromoxanthine was found to bind to oxidized enzyme and elicit the spectral change shown in Fig. 3A. The KD determined for the complex of oxidized enzyme with 8-bromoxanthine (Fig. 3A, inset) was 1.5 X M. This spectral change is, however, by no means specific for the interaction of 8-bromoxanthine with xanthine oxidase. Binding of salicylate to xanthine oxidase produces a spectral change almost identical concentration was on the order of t,he KO, it was necessary to determine the concentration of unbound inhibitor explicitly. This was calculated from the total inhibitor concentration minus the amount hound to enzyme (determined from the fractional absorbance change times the total enzyme concentration). This spectral change is dependent on the molybdenum center of the enzyme (see text).
with that shown in Fig. 3A on binding to native or cyanolyzed (inactivated) enzyme (data not sho4vn). Furthermore, binding of ligands to many simple flavoproteins exhibits the same or a very similar spectral change. Examples include the binding of aromatic amino acids to D-amino acid oxidase (21), pmethoxybenzaldehyde to old yellow enzyme (23), benzoate to D-amino acid oxidase (24), and tartronate to lactate oxidase (25). None of these enzymes are known to contain a cofactor other than flavin. It appears likely, therefore, that the ligandbinding phenomena associated with spectral changes similar to that observed with 8-bromoxanthine and oxidized xanthine oxidase reflect a change in the environment of the flavin cofactor and in the present case is not related to the molybdenum center of the enzyme. That this is the case is supported by two further pieces of evidence. First, uric acid, the product of enzymic action on xanthine and a compound known to interact at the molybdenum center (26,27) does not produce a spectral change similar to that shown in Fig. 3A (this work, data not shown). Second, inactivation of the molybdenum center by cyanide (11) does not alter the ability of 8-bromo-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from xanthine to elicit the spectral change, whereas removal of the FAD to generate the deflavo form of xanthine oxidase (18) completely abolishes the ability of the enzyme to produce the spectral change on addition of 8-bromoxanthine. Because inhibitor appears to be interacting with the flavin site of oxidized enzyme and does so with a KD several-fold greater than the Ki determined from the steady state and reductive half-reaction experiments, it is concluded that the interaction of 8-bromoxanthine with oxidized xanthine oxidase is not the interaction responsible for inhibition.
Binding of 8-bromoxanthine to dithionite-reduced xanthine oxidase, on the other hand, produces a completely different spectral change than that observed with oxidized enzyme, as shown in Fig. 3B. The KO associated with this spectral change is 18 PM (Fig. 3B, inset), two orders of magnitude lower than the K O observed for binding to oxidized enzyme. The extinction change at 360 nm for 8-bromoxanthine binding to reduced xanthine oxidase is 1.55 m"' cm-'. This value may be compared with the extinction change for alloxanthine binding to the reduced molybdenum center, which is approximately 4 In"' cm" at 380 nm (20). In contrast to the case with oxidized enzyme, the spectral change associated with the binding of 8-bromoxanthine to reduced xanthine oxidase is unperturbed on removal of the flavin but is abolished by pretreatment of the enzyme with cyanide (data not shown). The binding interaction associated with the spectral change shown in Fig. 3B thus appears to involve the molybdenum center of the enzyme, not the flavin, and is, therefore, the more likely candidate for the interaction giving rise to inhibition.
Kinetics of 8-Bromoxanthine Binding to Reduced Xanthine Oxidase-Given the convenient spectral change associated with the binding of 8-bromoxanthine to xanthine oxidase, the kinetic behavior of inhibitor binding could be examined with a stopped flow apparatus by monitoring the absorption increase at 360 nm. At all concentrations of 8-bromoxanthine employed, the reaction time course was accurately represented as a single exponential process. A plot of kobs versus the 8bromoxanthine concentration was apparently hyperbolic (not shown), but when replotted as l/kob, versus 1/(8-bromoxanthine) the data did not yield a straight line but rather was bowed, as shown in Fig. 4A. The most straightforward interpretation for this type of behavior is that inhibitor binding is a two-step equilibrium process, the spectral change being associated with the second step, with a finite rate for the reverse rate of the second step, k4 (28). plot indicates that the line drawn in Fig. 4A does in fact accurately represent the limiting slope of the data at low values of l/(Z). The equilibrium constant for the second step in the binding scheme, k4/k3, can be calculated and is found to be 0.11, i.e. the equilibrium position lies to the right as drawn above.
Interaction of Uric Acid and Xanthine with Reduced Xanthine Oxidase-In order to establish whether the interaction of 8-bromoxanthine is relevant to catalysis, titrations of reduced xanthine oxidase were performed with uric acid and xanthine. Uric acid has been reported to bind to reduced enzyme and raise the oxidation-reduction potential of the molybdenum MO"/MO'~ couple significantly (26). Xanthine also appears to raise the potentials of both MoV'/MoV and Mo"/MoIV couples, at least in the special case of enzyme that has been complexed with the inhibitor arsenite (15,29). Reduction of the arsenite-complexed molybdenum center to the MoV valence state (as observed by EPR) occurs earlier in the course of reductive titrations when xanthine is present than when it is absent. If the effect of 8-bromoxanthine on reduced enzyme could be correlated with observations using uric acid and xanthine, results with the inhibitor could prove relevant to the catalytic sequence. Titrations of reduced xanthine oxidase with uric acid and xanthine are shown in Fig.  5 , A and B, respectively. In both cases the spectral change observed on binding purine is very similar to that observed with 8-bromoxanthine (Fig. 3B). Furthermore, the K D values observed for urate and xanthine (18 and 25 PM; Fig. 5, insets) are comparable to those observed for 8-bromoxanthine (ie. 18 PM). The kinetics of urate and xanthine binding to dithionite-reduced enzyme is also similar to the results with 8bromoxanthine, giving linear double reciprocal plots with non-zero y axis intercepts (data not shown) to suggest again a pre-equilibrium step. The KO and k3 determined for urate were 115 PM and 130 s-', respectively, and 125 PM and 230 s-', respectively, for xanthine. From the x intercept of the double reciprocal plot (B, inset) a KD of 25 p M was determined. The extinction changes at 360 nm on binding urate and xanthine were 1.6 mM" cm" and 0.31 cm", respectively. In both titrations the spectral changes shown were corrected for dilution after addition of purine. The free ligand concentrations for the double reciprocal plots were calculated as described in the legend to Fig. 3.
The above results suggest that 8-bromoxanthine, uric acid and xanthine interact with the reduced molybdenum center in fundamentally the same way. Further evidence that this is the case comes from EPR work monitoring the paramagnetic MoV state at liquid nitrogen temperatures. The molybdenum EPR signal typically observed from the (functional) active site in enzyme partially reduced by sodium dithionite is shown in Fig. 6A. When the sample is reduced in the presence of 1.0 mM &bromoxanthine, the spectrum shown in Fig. 6B is observed. This spectrum is very similar to the so-called Rapid Type 2 signals observed with partially reduced enzyme in the presence of either xanthine or uric acid The enzyme concentration was on the order of 75 FM and was 70% active. Samples were reduced with sodium dithionite and in the latter case made 1.0 mM in 8-bromoxanthine prior to freezing in liquid nitrogen. The signal observed in the presence of 8bromoxanthine is similar to the Rapid Type 2 signal observed in the presence of xanthine (26). tion midpoint potential of that site. This is known to be the case with urate, which raises the midpoint potential of the molybdenum center by approximately 45 mV (27). These results were obtained at liquid nitrogen temperatures, however, and it has been shown that the various oxidationreduction potentials of xanthine oxidase are extremely sensitive to experimental conditions (temperature, pH, and buffer, Refs. 13 and 30). In order to determine the effect of 8bromoxanthine as well as urate on the oxidation-reduction potentials of the molybdenum center at room temperature under the present experimental conditions, potentiometric titrations of enzyme in the presence of 8-bromoxanthine or urate were performed using an apparatus similar to that described by Porras and Palmer (13), with 0.1 M pyrophosphate, pH 8.5, 0.3 mM EDTA as buffer. The same is true with 8-bromoxanthine, although the effect appears to be more pronounced on both potentials by some 10-20 mV. Given the uncertainty in integrating the EPR signals in these two titrations, this difference is not deemed significant. The essential point is that 8-bromoxanthine is seen to perturb the oxidation-reduction potentials of the (active) molybdenum center of xanthine oxidase in a manner very similar to that observed with urate.
Using the values determined for the molybdenum oxidation-reduction potentials in the absence and presence of 8bromoxanthine and the KD determined for inhibitor binding to reduced xanthine oxidase (Mol'), thermodynamic cycles may be constructed that permit the calculation of KD values for inhibitor binding to MoV' and Mo" valence states. The equilibrium constants thus obtained are 1.5 mM and 0.9 mM for 8-bromoxanthine binding to MoV1 and MoV, respectively. Two observations are noteworthy in regard to these values. First, the results indicate that the change in the chemistry of the molybdenum center that occurs on reduction of the metal (as reflected in the increased affinity for inhibitor on reduction) is associated for the most part with the MoV/ Mo" couple. Thus, at least in regard to the binding of 8bromoxanthine (but also urate) MoV more resembles MoV1 in its chemistry than it does MolV. Second, the K D calculated for binding of 8-bromoxanthine to MoV1 is the same as that determined experimentally for inhibitor binding to oxidized enzyme, monitoring a spectral change that is clearly due to a perturbation of the flavin environment. It is possible that this is merely a coincidence with two separate binding events (one at the molybdenum center, the other at the FAD) having the same KO, but we cannot at present rule out the possibility that inhibitor binding to oxidized enzyme occurs exclusively a the molybdenum center (albeit in a way that cannot involve the essential sulfur of the active site as binding to oxidized desulfoenzyme also gives the spectral change) and perturbs the flavin environment by means of a conformational change.

CONCLUSIONS
The substrate analog 8-bromoxanthine has been found to be an inhibitor of xanthine oxidase, having a Ki determined from steady state analysis of approximately 400 pM. Inhibition is uncompetitive with respect to xanthine and is found to be due to the preferential binding of inhibitor to the reduced form of the molybdenum center of enzyme (resulting in the inability of enzyme to participate in the reductive half-reaction of the catalytic cycle). This conclusion is supported by the observation of a rather tight KO for inhibitor binding to reduced enzyme (18 p~) determined from spectrophotometric titration and by the observation that inhibitor dramatically slows the anaerobic reduction of enzyme by xanthine but has only a minor effect on the reoxidation of reduced enzyme by molecular oxygen. The Ki for 8-bromoxanthine determined from the reductive half-reaction is 395 p~ in good agreement with the steady state results, but at variance with the KD of 18 p~ determined from equilibrium titration. This discrepancy is to be expected, however, if the inhibitory complex involves Mor". In the steady state and reductive half-reaction experiments the inhibitor interacts with only partially reduced enzyme species, and the fraction of molybdenum centers present as Mo" is much less than 1.0. The result is that a large fraction of the enzyme population does not have molybdenum in the appropriate oxidation state for complex formation, and the observed Ki will, therefore, be considerably higher than the KO for inhibitor binding to completely reduced enzyme (containing 100% Mol") as is observed.
Given the evidence that 8-bromoxanthine inhibits xanthine oxidase by binding to the reduced form of the molybdenum center, it might seem anomalous that the inhibitor would slow the rate of reduction of oxidized enzyme by xanthine. It must be remembered, however, that three substrate molecules are required to completely reduce xanthine oxidase and that the second and third xanthine molecules interact with enzyme that is already partially reduced. It is these two steps that are affected by inhibitor. Furthermore, although the intrinsic rate constants associated with the three steps in enzyme reduction must decrease in the order k, > kp > kg as the molybdenum center encountered by substrate becomes increasingly likely to become reduced, a semilogarithmic plot of the kinetic data appears to accelerate (31). This phenomenon tends to mask the slowing of the latter rates of reduction by 8-bromoxan-Inhibition of Xanthine Oxidase by 8-Bromoxanthine thine. Thus, while a rapid phase followed by a slow phase in the kinetic timecourse might be expected a priori in the presence of 8-bromoxanthine (as the second and third steps in reduction are appreciably slowed compared to the first step) this is not observed.' It is found that 8-bromoxanthine, urate, and xanthine all bind to reduced enzyme with similar Kn values and produce similar small but reproducible spectral changes. Furthermore, the kinetics of binding for each of these purines to reduced enzyme is similar, indicating in each case a pre-equilibrium step prior to formation of the complex giving rise to the spectral change. Urate and 8-bromoxanthine both perturb the oxidation-reduction potentials of the molybdenum center and in much the same way. It seems likely, therefore, that xanthine also perturbs the molybdenum potentials. Suggestive evidence that this is the case has been reported by Bray and co-workers (26) who performed potentiometric titrations with the similar enzyme xanthine dehydrogenase using xanthine as a reductant. If xanthine does in fact increase the potentials of the molybdenum center, the excess substrate inhibition of enzyme long observed with xanthine (34,35) could be readily explained. At higher xanthine concentrations, the level of reduced enzyme in the steady state increases, making it more likely that enzyme will be a t least partially reduced when it encounters a substrate molecule. According to the argument, on formation of the Michaelis complex, the molybdenum potentials would be raised. With electrons already present in the enzyme at the FAD and/or iron-sulfur centers (which are known to be in a very rapid oxidation-reduction equilibrium with the molybdenum center; Ref. 32), however, the possibility exists for the molybdenum to take up electrons not from substrate but from other components of the enzyme. The net result would be a Mo".xanthine complex unable to participate in the reductive half-reaction until the pair of electrons on the molybdenum were removed, either due to the equilibrium with the other sites on the enzyme or in the course of reoxidation of enzyme by molecular oxygen. This explanation for the observed excess substrate inhibition is consistent with the observation that inhibition is less pronounced a t higher oxygen concentrations, where the steady state level of reduced enzyme is lower (32), making it less likely for the MolV. xanthine complex to form.
It is somewhat surprising that 8-bromoxanthine, urate, and xanthine would bind to reduced xanthine oxidase in the same way, as is apparently the case. The bulky bromine atom especially might be expected to exert considerable steric hindrance. It is conceivable that all three purines bind via the pyrimidine subnucleus in exerting their effect on the molybdenum center. This appears unlikely, however, on the basis of preliminary results of experiments using x-ray absorption spectroscopy' that places the bromine atom of 8-bromoxan-That the distribution of reducing equivalents at the microscopic level within xanthine oxidase can affect the macroscopic properties of the enzyme has been observed before in a comparative study of Vmax for xanthine oxidase containing a series of flavin analogs having a wide range of oxidation-reduction potentials (32). In this case, it was shown that the distribution of reducing equivalents between Mo and FAD, based on the relative oxidation-reduction potentials of the two sites and the assumption of a rapid equilibrium existing between them (31) could entirely account for the low specific activity observed with flavin derivatives of low potential. As in the present case, the reaction ( i e . turnover) was slowed by as much as a factor of 20 because only a small fraction of the total enzyme population had an electron distribution appropriate for catalysis (i.e. Mo"' and FADH, as opposed to Mo'" and FAD). This type of phenomenon has been discussed in a recent review (33).
S. P. Cramer and R. Hille, unpublished results. thine approximately 4 A from the molybdenum atom, far too close for binding to be taking place via the pyridmidine subnucleus. One is left with binding via the imidazole subnucleus, with the likelihood of an intervening atom to explain the long Mo-Br distance.
Given that the Mo". urate complex is almost certainly a catalytic intermediate (in fact the most accessible intermediate, experimentally, after E,, and E r e d ) it is worth noting that 8-bromoxanthine is a more appropriate analog of product than of substrate. In this light the optical absorbance change associated with purine binding and particularly the ability to discern bromine in the extended x-ray absorption fine structure analysis of molybdenum provide the opportunity to gain important new insight into the interaction of purines with the active site of xanthine oxidase as well as the structure of the molybdenum center.