Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys535 and Cys992 residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4′-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4′-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD+ binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4′-dithiodipyridine, indicating that Cys1316 and Cys1324 are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation. Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys535 and Cys992 residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4′-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4′-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD+ binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4′-dithiodipyridine, indicating that Cys1316 and Cys1324 are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation. Xanthine oxidoreductase (XOR), 1The abbreviations used are: XOR, xanthine oxidoreductase; XO, xanthine oxidase; XDH, xanthine dehydrogenase; DTT, dithiothreitol; FDNB; fluorodinitrobenzene; 4,4′-DTPY, 4,4′-dithiodipyridine; MB, methylene blue; FAD, flavin adenine dinucleotide. xanthine dehydrogenase (XDH, EC 1.1.1.204), or xanthine oxidase (XO, EC 1.2.3.2) is a complex metalloflavoenzyme that catalyzes oxidation of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of NAD+ or molecular oxygen. The enzyme is a homodimeric protein of Mr 300,000 and is composed of independent subunits; each subunit contains one molybdopterin, two non-identical iron sulfur centers ([2Fe-2S] clusters), and one FAD (1Bray R.C. Boyer P.D. 3rd Ed. The Enzymes. XII. Academic Press, New York1975: 299-419Google Scholar, 2Hille R. Nishino T. FASEB J. 1995; 9: 995-1003Crossref PubMed Scopus (383) Google Scholar, 3Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1480) Google Scholar). The oxidative hydroxylation of xanthine to uric acid takes place at the molybdenum center, and reducing equivalents thus introduced are transferred rapidly via two iron sulfur centers to FAD, where physiological oxidation occurs (4Olson J.S. Ballou D.P. Palmer G. Massey V. J. Biol. Chem. 1974; 249: 4363-4382Abstract Full Text PDF PubMed Google Scholar). The mammalian enzymes exist in the NAD+-dependent form (xanthine dehydrogenase, XDH) in freshly prepared samples from organs under normal conditions, i.e. they exhibit low xanthine/O2 reductase activity but high xanthine/NAD+ reductase activity, even in the presence of O2 (5Della Corte E. Stripe F. Biochem. J. 1968; 108: 349-351Crossref PubMed Scopus (92) Google Scholar, 6Stripe F. Della Corte E. J. Biol. Chem. 1969; 244: 3855-3863Abstract Full Text PDF PubMed Google Scholar). XDH can be converted reversibly to xanthine oxidase (XO) by oxidation of cysteine residues or irreversibly by limited proteolysis (5Della Corte E. Stripe F. Biochem. J. 1968; 108: 349-351Crossref PubMed Scopus (92) Google Scholar, 6Stripe F. Della Corte E. J. Biol. Chem. 1969; 244: 3855-3863Abstract Full Text PDF PubMed Google Scholar, 7Della Corte E. Stripe F. Biochem. J. 1972; 126: 739-745Crossref PubMed Scopus (351) Google Scholar, 8Waud W.R. Rajagopalan K.V. Arch. Biochem. Biophys. 1976; 172: 354-364Crossref PubMed Scopus (174) Google Scholar, 9Nakamura M. Yamazaki I. J. Biochem. 1982; 92: 1279-1286Crossref PubMed Scopus (57) Google Scholar, 10Saito T. Nishino T. J. Biol. Chem. 1989; 264: 10015-10022Abstract Full Text PDF PubMed Google Scholar, 11Hunt J. Massey V. J. Biol. Chem. 1992; 267: 21476-21485Abstract Full Text PDF Google Scholar, 12Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar, 13Nishino T. Nishino T. J. Biol. Chem. 1997; 272: 29859-29864Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). XO has high reactivity toward O2 but negligible reactivity toward NAD+. As XO can reduce molecular oxygen to superoxide and hydrogen peroxide (1Bray R.C. Boyer P.D. 3rd Ed. The Enzymes. XII. Academic Press, New York1975: 299-419Google Scholar), XO is thought to be one of the key enzymes producing reactive oxygen species (14McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar). The crystal structures of bovine milk XDH and proteolytically produced XO have been solved and showed large conformational differences around the FAD (15Enroth T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google Scholar). Although the transition seems to occur in a similar way, whether caused by cysteine modification or proteolysis, the identification of the responsible cysteine residues is still a matter of controversy. It is not easy to identify the responsible residues, because the enzyme contains as many as 36 cysteine residues/monomer of rat XOR (12Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar), and many residues are modified by common cysteine-modifying reagents, such as 5,5′-dithiobis(nitrobenzoic acid) or iodoacetamide (16Saito T. Yokohama Med. Bull. 1987; 38: 151-168Google Scholar). It was, however, reported that only four cysteine residues were modified during the conversion from rat liver XDH to XO by titration with 4,4′-dithiodipyridine (4,4′-DTPY) (16Saito T. Yokohama Med. Bull. 1987; 38: 151-168Google Scholar). During the titration, two disulfide bonds were suggested to be formed by modification with 4,4′-DTPY, because the addition of 2 mol of 4,4′-DTPY stoichiometrically provides 4 mol of 4-thiopyridone. A similar observation was made by Hunt and Massey (11Hunt J. Massey V. J. Biol. Chem. 1992; 267: 21476-21485Abstract Full Text PDF Google Scholar) with the bovine milk enzyme. Although only four cysteine residues were modified during conversion from XDH to XO by this reagent, it was still difficult to identify the residues, because the modifier was released from the residues involved as part of the reaction. We then reported that relatively few cysteine residues were modified upon chemical reaction with FDNB (13Nishino T. Nishino T. J. Biol. Chem. 1997; 272: 29859-29864Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). During the initial 10 min of reaction with FDNB, two specific cysteine residues, Cys535 and Cys992, were labeled, and these residues were suggested to be involved in the rapid conversion from XDH to XO. Recently, Rasmussen et al. (17Rasmussen J.T. Rasmussenn M.S. Petersen T.E. J. Dairy Sci. 2000; 83: 499-506Abstract Full Text PDF PubMed Scopus (27) Google Scholar) reported the sulfhydryl group modification of bovine milk XOR using radioactive iodoacetic acid, and their results are consistent with our assignment. On the other hand, this interpretation has subsequently been challenged in a report describing gel analyses of the proteolytically cleaved disulfide form of XO (18McManaman J.L. Bain D.L. J. Biol. Chem. 2002; 277: 21261-21268Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The crystal structure of bovine XOR shows that Cys992 is situated on the surface of the molecule, but Cys535 seems to be located in the long linker peptide between the FAD and the molybdopterin domains, although the residue is not visible in the crystal structure most probably due to its flexibility. The site is on the linker on analyses of crystal structures of XDH and XO, as as et al. Y. Nishino T. Okamoto K. T. Eger B.T. Pai Nishino T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that the acid of to rat and in the bovine enzyme at the of a that of the linker peptide caused by cysteine oxidation or to the site The of the site is to be the of the in chemical between XDH and XO (15Enroth T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google Scholar). the present we two cysteine one of is responsible for rapid and the other for slow conversion from XDH to XO. We at the crystal structure of a rat mutant XOR in the form and that its polypeptide the XDH conformation. and wild-type for wild-type XOR were from T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). for mutant was from The was by Palmer of The and modification enzymes were from or New The or and were from The used in these was in our purified rat liver XOR, as (12Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar). was from was from and NAD+ was from other were of and M. and G. E. A of for and Scholar) was for the of the wild-type and mutant XOR The was using a in and a of the were using a was with the of rat liver XOR, and this was in the with in the presence of and to The were by and was with and by The was to the XOR for C535A by and for C992R by was to and introduced E. The were and with and for the C535A mutant and and for the C992R was with that been with the The mutant C535A/C992R was by of the C535A at the and at the As the mutants C535A/C992R/C1324S and the mutant was by using the XOR and the XOR was reported (13Nishino T. Nishino T. J. Biol. Chem. 1997; 272: 29859-29864Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), by and at the and The mutant was with the for by using a and of The each mutant enzyme was with and The of the was by The contains the and can be using as a T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar, M. and G. E. A of for and Scholar, J. M. T. G. J. 1990; PubMed Google Scholar). of wild-type recombinant was prepared as T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). with and was using a and of the recombinant was by to the T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar, M. and G. E. A of for and Scholar). identify were with of the and were We prepared a with a of of XOR and the of and were the as T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). of were as T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google the recombinant and were by T. Nishino T. K. PubMed Scopus Google Scholar, T. Nishino T. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). The enzymes were and with or 10 for at to xanthine dehydrogenase of XOR, by gel to DTT, as was as by PubMed Scopus Google Scholar) using from of a of recombinant were at in in a of Xanthine oxidoreductase with were determined by the as O2 and NAD+ or activity was determined by the at with in the presence of methylene XOR was determined from the at using of J.L. W.R. Rajagopalan K.V. J. Biol. Chem. 1974; 249: Full Text PDF PubMed Google Scholar) for the enzyme. XO was converted to XDH by incubation with or 10 at for The dehydrogenase to oxidase as by and Rajagopalan W.R. Rajagopalan K.V. Arch. Biochem. Biophys. 1976; 172: 354-364Crossref PubMed Scopus (174) Google Scholar) was determined as the of the at under in the presence of NAD+ to that in the of NAD+. to flavin (1Bray R.C. Boyer P.D. 3rd Ed. The Enzymes. XII. Academic Press, New York1975: 299-419Google Scholar) was by the in at in the presence of NAD+ under by the at of the enzyme used in the at were with a and of the C535A/C992R/C1324S of enzyme used for was purified on a gel of to enzyme As the recombinant XOR was in T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar), and the enzyme used for was the The enzyme was to in A T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar) and with for min at of the mutant XDH were by a of of protein and of DTT, and of of the enzyme were with their as a and in were at at a of K. of and a were and of of reported that two cysteine residues, Cys535 and Cys992, were involved in the conversion from XDH to XO, on the results of chemical modification with the of these residues, we mutants of rat XOR, and the be converted to XO by the residue Cys992 is by whereas the residue Cys535 is A. Nishino T. Noda K. Y. Nishino T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The form of recombinant rat liver XOR was in the T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). As the enzyme of it and T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). this we used the enzyme from the the demolybdo form, for analyses of and mutant in reduction where the demolybdo form not the results T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). the activity to flavin of XDH at was is with that of XOR purified from rat liver the T. Nishino T. K. PubMed Scopus Google Scholar). of the enzymes were in their XDH form and be as such in Although the wild-type XOR negligible XDH activity only for the of the mutant enzymes XDH activity even they XDH of the type, be to of these purified recombinant showed a of molecular at on although was a of in the C535A mutant The of the activity to flavin of these purified enzymes were between and to a form activity to flavin for enzyme is T. Nishino T. Arch. Biochem. Biophys. PubMed Scopus Google because of the presence of as in the of the enzyme T. Nishino T. J. Biol. Chem. 1989; 264: 10015-10022Abstract Full Text PDF PubMed Google Scholar). The of these were the at FAD not suggesting that the FAD binding site is the purified and mutants were similar in other conversion of and and C992R with purified recombinant wild-type and mutant XOR enzymes were with to to the XDH type, by gel to DTT, and the were with A is in the recombinant wild-type XOR and C535A/C992R mutant The mutant C535A and C992R showed similar shows the of cysteine residues during enzymes were converted readily to XDH by during min of incubation the of recombinant wild-type XDH, the XO activity was from to min by with 4,4′-DTPY, as in The activity determined in the presence of NAD+ at the initial during the reaction. On the other hand, the conversion from XDH to XO of the mutant was significantly slower that of wild-type with 4,4′-DTPY, although even the mutant was still converted slowly to the oxidase form during prolonged the oxidase activity of C535A/C992R from to min at to conversion from XDH to XO was in the mutants C535A and although the mutant C535A/C992R was the most resistant results although Cys535 and Cys992 are involved in rapid conversion by with 4,4′-DTPY, the conversion from XDH to XO not only Cys535 and Cys992, but other It be that the oxidase reaction with 4,4′-DTPY were to the XDH by incubation with 10 DTT, indicating that the conversion is due to modification of sulfhydryl residues of C535A/C992R of with and of by the of activity with of XO activity in a similar to the activity, suggesting that the conversion is to binding the binding of the C535A/C992R mutants with NADH, enzymes with of were and reduction were by with under As in the of by was to activity, suggesting that other cysteine residues exist near the NAD+ binding or C535A/C992R mutant was with 4,4′-DTPY in the presence of As in the conversion from XDH to XO was completely and the mutant activity even min that the other cysteine couple near the NAD+ binding site and can be from modification by the binding of the crystal structure of bovine XDH not XO (15Enroth T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google the peptide is the toward the NAD+ binding cysteine residues that form a disulfide are located near the the of these residues, we two C535A/C992R/C1316S and the the residue Cys1324 is by whereas the residue Cys1316 is A. Nishino T. Noda K. Y. Nishino T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). C535A/C992R/C1316S and C535A/C992R/C1324S XOR the mutant C535A/C992R XOR was in its XDH form, but it converted to the of XO activity during the On the other hand, mutants C535A/C992R/C1316S and C535A/C992R/C1324S were as XDH but their activity was not a of 10 to they were not converted to XO enzymes even a shows the of purified C535A/C992R/C1316S and C535A/C992R/C1324S and reduction with under The reduction min was the as in the of rat liver Although we the with C535A/C992R/C1316S or C535A/C992R/C1324S XOR, the were from each they showed the of and at that are of the XDH the reaction of the mutants C535A/C992R/C1316S and C535A/C992R/C1324S with mutants showed activity at from XDH to they the initial dehydrogenase activity during the reaction results are consistent with the that these mutants are not converted to XO by sulfhydryl oxidation and the that Cys1316 and Cys1324 are involved in the slower conversion from XDH to XO by sulfhydryl of the C535A/C992R/C1324S of C535A/C992R/C1324S mutant of rat liver XOR in the demolybdo form was and were to The structure was determined by molecular and using molecular The contains one in to the bovine milk XDH, contains one however, the two structures are very similar the of domains, the the and the the (12Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar, T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google Scholar). The to the in bovine milk XDH, although the molybdopterin was in this mutant, as from the results of chemical T. Y. S. Y. Okamoto K. Nishino T. J. Biochem. 2002; PubMed Scopus Google Scholar). It is that the structure of the molybdopterin of this mutant is not from that of the form of bovine milk XDH, and the acid residues the molybdopterin are situated at very similar and in similar the for the molybdopterin Although the of the was not visible in the bovine milk XDH structure probably because of the the FAD and the molybdopterin was in this mutant even the of residues were relatively high with the of for enzyme a although still in this part of the polypeptide of the rat enzyme with the bovine one Y. Nishino T. Okamoto K. T. Eger B.T. Pai Nishino T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The C992R residue is located at the of a of the molybdopterin and C535A is part of the linker the FAD to the molybdopterin The between the of residue and that of residue is As we is a of that a by the of the for the transition and by a toward the FAD Y. Nishino T. Okamoto K. T. Eger B.T. Pai Nishino T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The crystal structure of the present rat liver XDH mutant contains this acid and the takes the as in bovine milk are consistent with the that the mutant is in the XDH Although the of was the FAD site of the in the bovine crystal structure (15Enroth T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google Scholar), that peptide with the NAD+ binding of the in the crystal structure of the rat enzyme The peptide with a seems to the binding of NAD+ to the XDH Although for the acid residues between was the to Cys1316 and from was in the present The of between Cys1316 and Cys1324 is and disulfide a large of the it is very that the of a disulfide between Cys1316 and Cys1324 in conformational of the with the of this seems to be for binding at the flavin The results of the to of Cys535 and Cys992 in the rapid conversion from xanthine dehydrogenase to oxidase. mutants C992R and C535A significantly conversion of XDH to XO by disulfide or by the sulfhydryl modifier As the mutant C535A/C992R was most resistant to the rapid conversion. with FDNB results in to conversion, but on prolonged incubation of the the activity not due to modification near the xanthine binding site (13Nishino T. Nishino T. J. Biol. Chem. 1997; 272: 29859-29864Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, T. K. R. Massey V. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar). The that mutants conversion that these residues and Bain (18McManaman J.L. Bain D.L. J. Biol. Chem. 2002; 277: 21261-21268Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) the of Cys992 and Cys535 in the XDH to XO conversion they to of the FAD and molybdopterin proteolysis of bovine XO. The gel that they however, is to during the cysteine residues are of the Cys992 residue is by the of a bovine XO was under conditions, the of a disulfide with T. E. F. K. T. the crystal structure of the rat liver XDH mutant in this the of residues and are a large of the linker peptide be for disulfide between Cys992 and the It be that the between the two residues for disulfide the peptide were to As in of bovine milk XOR by and analyses of the crystal structures of bovine milk XDH and XO have the presence of a acid as a and a during the transition between the dehydrogenase and oxidase of XOR Y. Nishino T. Okamoto K. T. Eger B.T. Pai Nishino T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). between the acid residues of the are for of the XDH form of the enzyme. The residues at the of a that of the linker caused by cysteine oxidation or to the site enzyme in a in the of the to the of from the probably the for the can be caused by a in by disulfide between Cys535 and Cys992 or by proteolysis the linker the XDH, be converted to oxidase form, the linker peptide is because the flavin and molybdenum in The of the R. enzyme seems to be in the XDH form by S. M. G. PubMed Scopus Google Scholar, K. S. R. Rajagopalan K.V. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the C535A/C992R mutant not the rapid XDH to XO conversion with wild-type it was converted slowly by suggesting the of another cysteine couple responsible for the slower conversion. with of the C535A/C992R dehydrogenase as a of prolonged suggested that the other cysteine couple be to the binding site The that the slow conversion reaction of XDH to XO with 4,4′-DTPY was completely blocked in the presence of such The crystal structure of the bovine milk enzyme suggested that Cys1316 and Cys1324 were the most for such a The results of chemical modification with radioactive FDNB showed that of was We produced the mutant in a Although the protein was in its XDH form, it been converted to its oxidase form by the of the XDH form of the mutant was converted to the XO enzyme with 4,4′-DTPY under the as it is not that the mutant still has the to because the of cysteine modification at the can be by the rapid conversion because of disulfide between Cys535 and this two C535A/C992R/C1316S and were completely in their XDH in and they in this form the these two mutants with 4,4′-DTPY for min not conversion, and the and the of XDH and XO were the crystal the Cys1316 and residues are located to the and the between their is The peptide is the in the of the NAD+ binding and with the that seems to be for binding the NAD+ observation by the crystal structure of the complex of the bovine The differences in by this peptide in bovine XO, binding to a in the crystal in bovine XDH (15Enroth T. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F Proc. Natl. Acad. Sci. U. S. A. 2000; : 10723-10728Crossref PubMed Scopus (593) Google Scholar) and the binding site of the subunit in rat XDH) are by the the of bovine XO, can be whereas in the bovine XDH it toward the molecule, although with and in rat XDH, the binding site on the is results are consistent with a large of of this it in and of the binding its it is easy to that the part of the XOR a in to the of the binding of a disulfide in the of NAD+ interpretation of the of the peptide is not only on our but is by our that the slow XDH to XO conversion reaction of C535A/C992R with 4,4′-DTPY was completely blocked in the presence of and that the XO form, was probably by disulfide between the two cysteine residues, be by The that the of acid residues between and was not in the crystal structure that this of the is and be to to disulfide between Cys1316 and are under to of the form of XO, a form that contains the to a of conformational near the flavin are caused by this oxidative with

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