The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus*

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, kred (the limiting rate of enzyme reduction by substrate at high [S]) decreased ∼20-fold in an R310K variant and 2 × 104-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed ∼4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in kred greater than 103), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in kred less than 50-fold). The effect was such that the 4000-fold range in kred seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which “good” substrates are bound “correctly” in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, “poor” substrates bound upside down relative to this “correct” orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k red (the limiting rate of enzyme reduction by substrate at high [S]) decreased ϳ20-fold in an R310K variant and 2 ؋ 10 4 -fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed ϳ4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k red greater than 10 3 ), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k red less than 50-fold). The effect was such that the 4000-fold range in k red seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.
The molybdenum-containing hydroxylases represent a unique solution to the hydroxylation of carbon centers. Other monooxygenases introduce an oxygen atom derived from O 2 and consume two reducing equivalents (along with the two removed from the substrate to be hydroxylated) in reducing O 2 to water. The molybdenum enzymes, on the other hand, utilize water itself as the ultimate source of the oxygen atom incorporated into product. In the case of enzymes such as xanthine dehydrogenase, not only are molybdenum enzyme reducing equivalents not consumed in carrying out the catalyzed reaction, but in fact physiologically useful reducing equivalents in the form of NADH are generated. As such, these enzymes represent a unique solution to the chemistry of hydroxylation, and the requisite cleavage of a carbon-hydrogen bond that accompanies it (1).
The xanthine dehydrogenase from Rhodobacter capsulatus is an (␣␤) 2 heterotetramer comprising two copies each of the XdhA and XdhB gene products (2). XdhA possesses two different [2Fe-2S] iron-sulfur clusters of the spinach ferredoxin variety as well as FAD, with each redox-active center located in a separately folded and contiguous domain of the polypeptide. XdhB possesses a molybdenum center with a square pyramidal LMo VI OS(OH) coordination sphere, with L representing a unique pyranopterin cofactor coordinated to the metal via an enedithiolate sidechain. This organic cofactor is common to all molybdenum-and tungsten-containing enzymes, with the sole exception of the multinuclear molybdenum-and iron-containing active site of nitrogenase (1). The structure of the R. capsulatus enzyme is known (3), and it bears strong structural as well as sequence homology to other members of the molybdenum hydroxylase family of enzymes (including bovine xanthine oxidoreductase (4, 5) and quinoline 2-oxidoreductase from Pseudomonas putida (6)). The structure of the active site of the R. capsulatus xanthine dehydrogenase is shown in Fig. 1.
The molybdenum hydroxylases, which also include aldehyde oxidases from vertebrate, plant, and bacterial sources, are thought to share a common reaction mechanism, which has been best worked out in the case of the bovine enzyme (7)(8)(9). Catalysis is initiated by abstraction of a proton from the Mo-OH group by a universally conserved active site glutamate residue (7) followed by nucleophilic attack on the carbon center to be hydroxylated and concomitant hydride transfer to the MoϭS of the molybdenum center. This reaction yields an LMo IV (SH)(OR) intermediate, with OR representing the now hydroxylated product coordinated to the molybdenum via the newly introduced hydroxyl group. The catalytic sequence is completed by displacement of the bound product from the molybdenum coordination sphere by hydroxide from solvent, electron transfer out of the molybdenum center to the FAD (via the [2Fe-2S] centers), and deprotonation of the Mo-SH to give the original, oxidized LMoOS(OH) form of the center. The specific sequence of these latter events varies with the substrate used and the reaction conditions, but the LMo IV (SH)(OR) species is considered an obligatory intermediate.
In addition to the glutamate residue thought to act as a general base, another highly conserved residue in the active site of the xanthine-utilizing enzymes (but not the aldehyde-utilizing ones) is Arg-310 (Arg-880 in the bovine enzyme) (Fig. 1). This residue lies some 10 Å from the molybdenum center, too far to participate directly in catalysis. In the structure of the enzyme with the mechanism-based inhibitor alloxanthine, however, Arg-310 is hydrogen-bonded to the back side of the heterocycle via one of the carbonyl groups of the latter (3). Also, in a model of urate binding to reduced enzyme based on the crystal structure of the aldehyde oxidoreductase from Desulfovibrio gigas (7), the equivalent Arg is suggested to interact similarly with bound product. In the present work, we examined the catalytic role of Arg-310 and found, surprisingly, that its exchange to methionine results in a 20000-fold decrease in k red (the limiting rate of enzyme reduction upon mixing with substrate). Thus, ϳ4.5 kcal/mol of the free energy available to the enzyme from its interaction with substrate is used to accelerate reaction rate rather than to stabilize the E⅐S complex. An analysis of the reaction of wild-type and R310M or R310K enzymes with a series of other purine substrates provides information on how substrate orients itself in the active site for effective catalysis.

EXPERIMENTAL PROCEDURES
Protein Purification-Recombinant R. capsulatus xanthine dehydrogenase was purified using the procedure described by Leimkühler et al. (11), with affinity chromatography on a Sepharose 4B/folate gel as the final step. By using PCR mutagenesis, amino acid exchanges R310M and R310K were introduced into R. capsulatus xanthine dehydrogenase. The generated variants were expressed under the same conditions as the wild-type enzyme and purified by nickel-nitrilotriacetate chromatography, Q-Sepharose, and size exclusion chromatography. The purified enzymes were concentrated by ultrafiltration, gel-filtered using a PD-10 gel filtration column (GE Healthcare), equilibrated with 50 mM Tris, 1 mM EDTA, 2.5 mM DTT, pH 7.5, and stored at Ϫ70°C until used. The iron content of wildtype xanthine dehydrogenase and the R310M and R310K variants was determined to be in a range of 93 to 95%, whereas the molybdenum content varied for the proteins: 99% for wild-type xanthine dehydrogenase, 67% for the R310M variant, and 50% for the R310K variant, as analyzed by ICP-OES (Perkin Elmer Optima, DV2100). The levels of molybdenum saturation correspond well with the amount of the pterin cofactor present in the enzymes as determined by conversion to Form A described previously (11). The wild-type enzyme was determined to be approximately 80% active.
Enzyme Assays and Rapid Reaction Kinetics-Routine enzyme assays were carried out as described previously (10) at 25°C in 20 mM Tris, 0.2 mM EDTA, pH 7.8, monitoring the absorbance change at 340 nm due to reduction of NAD ϩ to NADH. The enzyme concentration was determined from the absorbance at 465 nm using an extinction coefficient of 31.6 mM Ϫ1 cm Ϫ1 (11). Reductive half-reaction experiments were performed under anaerobic conditions using an Applied Photophysics SX-18MV kinetic spectrophotometer with a 1-cm observation path length. Standard reaction conditions were 20 mM Tris, 0.2 mM EDTA, pH 7.8, at 4°C. In a typical experiment, enzyme at a concentration of 12-14 M was mixed with an equal volume of substrate solution, the latter at concentrations ranging from 20 M to 2.0 mM. The reaction was monitored at 460 and 620 nm over an appropriate time scale, and the observed kinetic transients fit to exponentials to obtain k obs . For those substrates for which k obs varied with substrate concentration, k obs was then plotted against substrate concentration to obtain k red , the limiting rate of reduction at high [S], and the dissociation constant K d . For those substrates (e.g. 2-hydroxy-6-methylpurine, as seen previously with the bovine enzyme (12)) for which k obs did not vary, the average of the observed values was taken as k red .
Tris hydrochloride was from Fisher Scientific. Xanthine, 1-methylxanthine, 2-thioxanthine, and 6-thioxanthine were from Sigma, 2,6-diaminopurine from Aldrich, and 2-hydroxy-6-methylpurine from the Sigma-Aldrich Library of Rare Chemicals. Other reagents were of the highest purity available commercially and were used without further purification.

RESULTS
Site-directed Mutagenesis of Arg-310 of R. capsulatus Xanthine Dehydrogenase-Both active site residues Glu-232 and Glu-730 in the R. capsulatus enzyme have been shown to be

Arg-310 in Xanthine Dehydrogenase from R. capsulatus
important for catalysis. Glu-730 is thought to act as an active site base in the initial step of the reaction, and its mutation to an alanine reduces the limiting rate of enzyme reduction by substrate by a factor of at least 10 7 . Glu-232 is involved in both substrate binding and transition state stabilization, as its mutation to alanine results in a 10-fold decrease in k red and a 10-fold increase in K d (10). To analyze the role of Arg-310 for R. capsulatus xanthine dehydrogenase, the variants R310K and R310M were generated as described under "Experimental Procedures." The absorption spectra of the purified variants were very similar to the wild-type enzyme, and the difference seen in the region around 320 nm is due to a rather lower MoϭS content of the mutated proteins ( Fig. 2A). The amino acid exchanges were chosen on the basis of amino acid alignment of xanthine-and aldehyde-utilizing enzymes, which showed that these proteins share an identity of 50%. There are some differences in the cofactor binding domain between xanthine-and aldehyde-utilizing enzymes, and the highly conserved residues Glu-232 and Arg-310 (R. capsulatus numbering) involved in substrate binding in the R. capsulatus enzyme are typically exchanged to valine and methionine, respectively, in aldehyde oxidases (Fig.  2B). The x-ray structure of R. capsulatus xanthine dehydrogenase with alloxanthine at the active site showed that Arg-310 forms a hydrogen bond with the oxygen atom of the 6-position of alloxanthine (corresponding to the 2-position of xanthine). Thus we wanted to investigate the effects of an exchange of arginine to lysine and methionine on substrate binding and determine the catalytic constants.
The Reaction of Wild-type R. capsulatus Xanthine Dehydrogenase and R310K/R310M Variants with Xanthine-The reductive half-reaction of wild-type R. capsulatus xanthine dehydrogenase exhibits a hyperbolic dependence of the observed rate constant on substrate concentration, yielding at 4°C values for k red and K d of 29 s Ϫ1 and 25 M, respectively, with a value for k red /K d of 1.2 ϫ 10 6 M Ϫ1 s Ϫ1 , in reasonable agreement with previous work (10). Upon substitution of Arg-310 with lysine, the corresponding values are 1.6 s Ϫ1 , 45 M and 3.7 ϫ 10 4 M Ϫ1 s Ϫ1 , indicating that this conservative mutation significantly compromises but does not abolish the catalytic power of the enzyme. Mutation to methionine, on the other hand, reduces k red to 0.0017 s Ϫ1 , a reduction of more than 10 4 from the value seen with the wild-type enzyme. In the case of the R310M variant, no substrate concentration dependence of the observed rate constant was seen over the range of 20 M to 2.0 mM, as has been observed previously with the slow substrate 2-hydroxy-6-methylpurine with bovine xanthine oxidase (12). This contrasting behavior of the wild-type enzyme and R310M variant notwithstanding, it is evident from a comparison of k red values alone that Arg-310 contributes ϳ4.5 kcal/mol toward transition state stabilization in the wild-type enzyme. As indicated in Table 1, the R310K variant was generally more active than the R310M variant for a variety of purine substrates, indicating that retaining the positive charge at position 310 gave rise to considerably less dramatic consequences on the kinetic behavior of the enzyme. It is thus evident that a positively charged residue at position 310 leads to significant transition state stabilization in the enzyme-catalyzed reaction.
The very great effect on k red upon mutating Arg-310 to methionine is surprising given that Arg-310 lies ϳ10 Å from the active site molybdenum and on the opposite end of the substrate from the site that is hydroxylated (implying that a significant amount of the free energy available to the system from the favorable interaction between substrate and Arg-310 is used to stabilize the transition state rather than simply bind to substrate). As seen in Fig. 3, however, a positive charge in the vicinity of the C-6 position is expected to resonance-stabilize the negative charge accumulating on the ring as a result of nucleophilic attack at C-8. As discussed further below, the fact that this stabilization occurs at C-6 rather than C-2 has implications regarding the orientation of substrate in the active site for productive catalysis.
The Reaction of Additional Purine Substrates with Wild-type Xanthine Dehydrogenase-In addition to xanthine, we also examined the reaction of R. capsulatus xanthine dehydrogenase with 1-methylxanthine, 2-thioxanthine, 6-thioxanthine, 2,6-diaminopurine, and 2-hydroxy-6-methylpurine, compounds for which the structures are shown in Fig. 4. These purines were selected taking into consideration that xanthine dehydrogenase is also able to hydroxylate at C-2 and C-6 as well as C-8, when these positions are available; each member of the present set is substituted in such a way that it can only be hydroxylated at the C-8 position (as is xanthine), and as such they constitute a proper homologous series in which to examine substrate specificity. The results of this kinetic study are tabulated in Table 1.
Both 2-and 6-thioxanthine were found to be effective substrates for the R. capsulatus xanthine dehydrogenase, with k red and K d values comparable with those seen with xanthine. These two thio derivatives have also previously been found to be effective substrates for bovine xanthine oxidase (13). By contrast, 1-methylxanthine, a good substrate for the bovine enzyme, exhibits a 10-fold reduced k red and 2-fold increased K d with the R. capsulatus enzyme. Although it is among the best substrates for the bovine enzyme (13), it is rather less effective than xanthine as a substrate for the R. capsulatus enzyme. On the other hand, 2-hydroxy-6-methylpurine and especially 2,6-diaminopurine are manifestly poorer substrates than xanthine (or either of the thio derivatives) for the wild-type R. capsulatus enzyme, with k red values of 0.17 s Ϫ1 and 0.007 s Ϫ1 , respectively.

The Reaction of Additional Purine Substrates with the R310K and R310M Variants of R. capsulatus Xanthine Dehydrogenase-
The trend seen with xanthine as substrate was generally observed for the other purines used in this study, with reactivity moderately reduced in the R310K variant and significantly reduced in the R310M variant. The exception was seen with 2-hydroxy-6-methylpurine, where the R310K variant was actually modestly more active than the wild-type enzyme, by a factor of ϳ4. Interestingly, there was a very small range of values for k red for the R310M variant with the substrates used, a factor of only 4 as compared with 4000 for wild-type enzyme (or 13,000 for the wild-type bovine enzyme (13)). Evi-dently, Arg-310 plays a significant role in substrate selectivity among the purines. Most of the substrates (excepting 2,6-diaminopurine) yielded a typical hyperbolic dependence of k obs versus [S] with wild-type enzyme, whereas most (excepting 6-thioxanthine) exhibited no substrate concentration dependence of the observed rate constant over the range of 20 M to 2.0 mM with the R310M variant.

DISCUSSION
On the basis of the experiments described above, it is evident that the substrates investigated herein fall broadly into two groups: members of the first are effective substrates of the wildtype enzyme and are hydroxylated significantly less effectively by the R310M variant, whereas members of the second are poor substrates of the wild-type enzyme but are much less affected by mutation of Arg-310 to methionine. Included in the first group is the physiological substrate xanthine, in which hydroxylation proceeds more than 10 4 -fold more slowly in the variant than in the wild-type enzyme. Given that Arg-310 is quite removed from the site where hydroxylation occurs, it is interesting that k red is affected to the extent that it is upon mutation of Arg-310 to methionine. It is evident that a considerable amount of the free energy available to the enzyme from the favorable interaction of this residue with substrate (some 4.5 kcal/mol) is used not to stabilize the E⅐S complex but instead to stabilize the transition state, thereby accelerating the reaction. How this likely occurs is illustrated in Fig. 3, where it can be seen that interaction of Arg-310 with the C-6 carbonyl group of substrate stabilizes negative charge accumulation on the heterocycle that accompanies nucleophilic attack at C-8.
Effective neutralization of negative charge accumulation on substrate in the course of the reaction is best achieved if substrate binds in the active site with the orientation shown in Fig.  3, with the C 6 ϭO carbonyl in a position to interact with Arg-310. In the crystal structure of the reduced R. capsulatus enzyme with the tight-binding inhibitor alloxanthine, however, it is the carbonyl equivalent to C 2 ϭO of xanthine (somewhat confusingly, the numbering convention of heterocycles is such that the equivalent carbonyl of alloxanthine is designated C 6 ϭO (14)) that interacts with Arg-310 (3). It is important to recognize, however, that in the complex with reduced enzyme, alloxanthine sits ϳ2 Å closer to the molybdenum than does nascent product in the course of the reaction, because alloxanthine coordinates directly to the molybdenum via N-8 of its pyrazole ring, whereas the C-8 position of nascent product is bridged to the metal via the hydroxyl oxygen that is introduced catalytically (Fig. 5). Being somewhat further removed from the molybdenum, it is possible that the catalytically preferred orientation of substrate is in fact inverted relative to the orientation seen crystallographically in the alloxanthine complex. Indeed, a model for urate bound to xanthine oxidoreductase based on the structure of the D. gigas aldehyde oxidoreductase (which, unusually among the aldehyde-utilizing enzymes, possesses Arg at position 504, equivalent to Arg-310 in the R. capsulatus enzyme) has Arg-504 interacting with the C 6 ϭO rather than C 2 ϭO carbonyl group of the docked product (7). 3 For these reasons, the orientation with C 6 ϭO inter-acting with Arg-310 more likely represents that of substrate at the outset of catalysis.
Considering now the two poorest substrates for the wild-type enzyme, 2,6-diaminopurine and 2-hydroxy-6-methylpurine, an examination of their structures indicates that neither will be able to interact effectively with Arg-310 via their 6-positions (occupied by an amino group and a methyl group, respectively). It is thus possible that the low reactivity of these nonphysiological substrates is due to their binding in an inverted orientation relative to xanthine. Were this the case, then the catalytic power deriving from Arg-310 would be lost, but as is in fact observed, there would be relatively little effect upon loss of the arginine upon mutation to methionine because the arginine was not contributing to catalysis in the first place. The data thus suggest that the difference in reactivity of the two groups of substrate is because of their different orientations in the active site.
An additional observation regarding the reductive half-reaction of the R310M variant is worth a brief mention. With each of the substrates shown in Fig. 4, the reaction with the R310M variant as followed at 460 nm exhibited an initial absorbance decrease followed by a rise before the subsequent loss of absorption that made up the majority of the absorbance change. The effect was most pronounced at high substrate concentrations, although the specific concentration at which this behavior became pronounced varied from one substrate to the next. The initial absorbance decrease was always only a very small fraction of the total absorbance change observed in the course of a given transient (less than 5%), yet was distinct enough to suggest the possibility of intermediate formation in the mechanism of this mutated form of xanthine dehydrogenase. This phenomenon is currently under further investigation.
In conclusion, in the present work we have demonstrated that the active site Arg-310 of R. capsulatus xanthine dehydrogenase plays an important role in catalysis, contributing ϳ4.5 kcal/mol toward transition state stabilization and accounting for a 2 ϫ 10 4 -fold increase in rate acceleration. Arg-310 is positioned in the active site well away from the molybdenum center and the C-8 position of substrate that is hydroxylated, but it apparently lowers the activation energy for the reaction by stabilizing negative charge accumulation on the heterocycle through an electrostatic interaction with the C 6 carbonyl oxygen of substrate. This in turn implies that substrate binds in the opposite orientation to that seen in the structure of the 3 In generating this model, it was necessary to exchange Phe-425 in the structure of the D. gigas enzyme to a Glu and to assume that the hydroxyl groups of Tyr-535 and Tyr-622, which are both phenylalanine in the xanthine-utilizing enzymes, do not alter the mode of substrate binding. It is further to be noted that Tyr-622 of the D. gigas enzyme, which was not shown in the original model (7), occupies a significantly different orientation relative to the molybdenum center than does the corresponding Phe-459 (Phe-1009 in bovine) in the subsequently determined R. capsulatus and bovine structures.  (reduced) enzyme in complex with the mechanism-based inhibitor alloxanthine (but like that seen in the model of urate bound to the D. gigas aldehyde oxidoreductase (7)). This conclusion is supported by the results of a kinetic study utilizing a homologous series of purines; those that are effective substrates of the wild-type enzyme, presumably binding in an orientation similar to xanthine and making use of the catalytic contribution of Arg-310, are strongly affected by the exchange of Arg-310 to methionine, whereas those that react most slowly with enzyme (2,6-diaminopurine and 2-hydroxy-6-methylpurine) have functional groups at position 6 (amino and methyl, respectively) that prevent interaction with Arg-310. We suggest that these substrates bind in an inverted orientation to that seen with xanthine, accounting for their low reactivity. Because these purines are not able to take advantage of transition state stabilization by Arg-310 in wild-type enzyme, they are expected to be less sensitive to its mutation to methionine, as was in fact observed. The disparity in reaction rate among the purine substrates used here with the R310M variant is 1000-fold less extensive than with the wild-type enzyme, indicating that Arg-310 plays a significant role in determining the substrate specificity of xanthine dehydrogenase. Finally, it is worth noting that the catalytic role for Arg-310 proposed here also provides an explanation as to why the arginine is not generally conserved in the aldehyde oxidases, because with these enzymes negative charge accumulation is expected to be restricted to the carbonyl oxygen of substrate, much nearer the molybdenum center.