Xanthine oxidoreductase from bovine milk can be prepared in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depending on the number of protein cysteines versus cystines. Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxygen, whereas XO only reacts significantly with oxygen. The preference for oxidizing substrate is partially explained by thermodynamics. Unlike XDH, the midpoint potential of the FAD, the center at which oxygen and NAD react, is too high in XO to efficiently reduce NAD (Hunt, J., Massey, V., Dunham, W.R., and Sands, R.H. (1993) J. Biol. Chem. 268, 18685–18691). To distinguish between changes in thermodynamics and in substrate binding, samples of both XO and XDH have been prepared in which the native FAD has been replaced with an FAD analog of different redox potential, 1-deaza-FAD or 8-CN-FAD. Reductive titrations indicate that both 1-deaza-XO and 1-deaza-XDH have a flavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XDH each have a flavin potential higher than XO. Both the low potential 1-deaza-XO and the high potential 8-CN-XDH contain essentially no xanthine/NAD activity. However, 1-deaza-XDH does exhibit xanthine/NAD activity, and 8-CN-XO has normal xanthine/oxygen activity.The binding of NAD to oxidized XO and XDH was investigated by ultrafiltration and isothermal titration calorimetry. TheK d for the binding of NAD to XDH was determined to be 280 ± 145 μm by ultrafiltration and 160 ± 40 μm by isothermal titration calorimetry. No evidence for the binding of NAD to XO by either method could be obtained. A low flavin midpoint potential is necessary but not sufficient for dehydrogenase activity. Xanthine oxidoreductase from bovine milk can be prepared in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depending on the number of protein cysteines versus cystines. Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxygen, whereas XO only reacts significantly with oxygen. The preference for oxidizing substrate is partially explained by thermodynamics. Unlike XDH, the midpoint potential of the FAD, the center at which oxygen and NAD react, is too high in XO to efficiently reduce NAD (Hunt, J., Massey, V., Dunham, W.R., and Sands, R.H. (1993) J. Biol. Chem. 268, 18685–18691). To distinguish between changes in thermodynamics and in substrate binding, samples of both XO and XDH have been prepared in which the native FAD has been replaced with an FAD analog of different redox potential, 1-deaza-FAD or 8-CN-FAD. Reductive titrations indicate that both 1-deaza-XO and 1-deaza-XDH have a flavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XDH each have a flavin potential higher than XO. Both the low potential 1-deaza-XO and the high potential 8-CN-XDH contain essentially no xanthine/NAD activity. However, 1-deaza-XDH does exhibit xanthine/NAD activity, and 8-CN-XO has normal xanthine/oxygen activity. The binding of NAD to oxidized XO and XDH was investigated by ultrafiltration and isothermal titration calorimetry. TheK d for the binding of NAD to XDH was determined to be 280 ± 145 μm by ultrafiltration and 160 ± 40 μm by isothermal titration calorimetry. No evidence for the binding of NAD to XO by either method could be obtained. A low flavin midpoint potential is necessary but not sufficient for dehydrogenase activity. Xanthine oxidoreductase catalyzes the oxidation of hypoxanthine and xanthine to urate and is involved in purine catabolism in mammals. Isolated from bovine milk, the enzyme exists as a dimer, containing one molybdopterin, one FAD, and two plant ferredoxin-type 2Fe/2S centers per 145-kDa subunit (1Kramer S.P. Johnson J.L. Ribeiro A.A. Millington D.S. Rajagopalan K.V. J. Biol. Chem. 1987; 262: 16357-16363Abstract Full Text PDF PubMed Google Scholar, 2Massey V. Brumby P.E. Komai H. Palmer G. J. Biol. Chem. 1969; 244: 1682-1691Abstract Full Text PDF PubMed Google Scholar, 3Palmer G. Massey V. J. Biol. Chem. 1969; 244: 2614-2620Abstract Full Text PDF PubMed Google Scholar, 4Avis P.G. Bergel F. Bray R.C. J. Chem. Soc. (Lond.). 1956; 253: 1219-1226Crossref Google Scholar). Reducing substrates such as xanthine react at the molybdenum center (5Tanner S.J. Bray R.C. Bergmann F. Biochem. Soc. Trans. 1978; 6: 1328-1330Crossref PubMed Scopus (30) Google Scholar), whereas oxidizing substrates such as oxygen or NAD react at the FAD (6Komai H. Massey V. Palmer G. J. Biol. Chem. 1969; 244: 1692-1700Abstract Full Text PDF PubMed Google Scholar, 7Nishino T. Nishino T. Schopfer L.M. Massey V. J. Biol. Chem. 1989; 264: 6075-6085Abstract Full Text PDF PubMed Google Scholar). Like other molybdenum hydroxylases, the oxygen incorporated into substrate is derived from water, and electron equivalents are released in the hydroxylation reaction. Electrons from xanthine are transferred either to NAD or to molecular oxygen, depending on the form of the enzyme present. Xanthine dehydrogenase-type (XDH) 1The abbreviations XDHxanthine dehydrogenaseXOxanthine oxidase2Fe/2Siron-sulfur center; fr. fraction enzyme prefers NAD as an electron acceptor (8Batteli M.G. Lorenzoni E. Stirpe F. Biochem. J. 1973; 131: 191-198Crossref PubMed Scopus (122) Google Scholar) but in the absence of NAD will catalyze xanthine/oxygen turnover at 30% the rate of xanthine/NAD turnover (9Harris C.M. Massey V. J. Biol. Chem. 1997; 272: 8370-8379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Xanthine oxidase-type (XO) enzyme only utilizes oxygen as its oxidizing substrate, to any significant extent. XDH from bovine milk can be converted to XO irreversibly by proteolysis (10Stirpe F. Della Corte E. J. Biol. Chem. 1969; 244: 3855-3863Abstract Full Text PDF PubMed Google Scholar, 11Amaya Y. Yamazaki K. Sato M. Noda K. Nishino T. Nishino T. J. Biol. Chem. 1990; 265: 14170-14175Abstract Full Text PDF PubMed Google Scholar) or reversibly by oxidation of cysteines to cystines (8Batteli M.G. Lorenzoni E. Stirpe F. Biochem. J. 1973; 131: 191-198Crossref PubMed Scopus (122) Google Scholar, 12Saito T. Yokohama Med. Bull. 1987; 38: 151-168Google Scholar). Approximately eight cysteines are oxidized to four cystines on converting milk XDH to XO (12Saito T. Yokohama Med. Bull. 1987; 38: 151-168Google Scholar). XDH is thought to be the form in T. Med. Biol. Scopus Google Scholar), and is evidence that oxygen from XDH or XO be in J. Google Scholar, J. Med. PubMed Scopus Google Scholar, M. T. PubMed Scopus Google Scholar). xanthine dehydrogenase xanthine oxidase center; fr. fraction is evidence for a between XDH and XO in the FAD binding XDH forms of the flavin to XO T. Nishino T. Massey V. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). XDH an to the FAD, as of the NAD analog of the of XDH T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and NAD only to XDH, no changes on the of either to oxidized XO. The redox potential of the in XDH, J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), is for the of at Scholar). However, the in Palmer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar), is too high for of the of the low redox potential of the and the of XDH in NAD as an oxidizing The midpoint potential of the was by of the normal FAD with FAD containing different redox the redox of the two enzyme forms, be to the of NAD as an oxidizing The low potential for the J. PubMed Scopus Google Scholar) was to an oxidase-type enzyme with a low midpoint of and Massey Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar) has that 1-deaza-XO has a low flavin potential of at the higher The has a redox potential of Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of 8-CN-XDH in a dehydrogenase-type enzyme with an to be too high for NAD The flavin of determined to the 2Fe/2S centers by titrations or by in the of redox of the xanthine/NAD and xanthine/oxygen to any changes in the oxidizing NAD binding have on changes of the enzyme on of NAD binding was by the of NAD in of NAD and oxidized XO or XDH and by isothermal calorimetry. that NAD binding to XDH and XO is but is not to in the of XO. in substrate binding and are XDH was by the method of and Massey J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). XO was in the but with the of The of 1-deaza-FAD has been by J. PubMed Scopus Google Scholar). The and of was by and Massey and has been Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). XDH samples for at with was by on a at in of XO prepared by the method of Komai (6Komai H. Massey V. Palmer G. J. Biol. Chem. 1969; 244: 1692-1700Abstract Full Text PDF PubMed Google Scholar). XDH was prepared in the but with the of at The and of than of of was by enzyme with a of the FAD for at on flavin was by and in a of XO and XDH with normal FAD that native could be of 1-deaza-FAD enzyme of oxygen in as (9Harris C.M. Massey V. J. Biol. Chem. 1997; 272: 8370-8379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). V. 1978; PubMed Scopus Google Scholar) by of enzyme in a containing and to of the enzyme titrations by of an from a into an of are as in which the at a 2Fe/2S is as a of the at a FAD of the determined to the 2Fe/2S centers by with from the the redox of 2Fe/2S and 2Fe/2S and the changes of the and FAD The fraction of each center was at a potential, E. changes by the of changes for each center by the at at FAD the changes are at the FAD The fraction at the 2Fe/2S was in the but with the of 2Fe/2S 2Fe/2S and as from the potential, and the T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, Scholar), of the The potential was at and method on the that the of the 2Fe/2S centers are by flavin The method is as the potential of the FAD is not too from the 2Fe/2S 2Fe/2S 2Fe/2S Palmer G. J. Biol. Chem. Full Text PDF PubMed Google XO was as in the of the at The of of the was from its at XO The of of the enzyme was at an for the of To the between the redox potential of and that of the the of the was the of the to the method of K. PubMed Google Scholar), a method for the of the redox of native XDH J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). samples of XDH containing an or of dehydrogenase μm of oxygen in an with by a of for The was To of the XDH by an μm and in from the The was in the and the was by at with as the the the of of the was by the in at the enzyme to than of the the and the of of the flavin was at which is for the the of of the was by the of in at the enzyme to than of the and the of of the flavin was from the in at the changes of the the of of the was by the in at changes with and of the flavin of 8-CN-XDH are changes at with the 2Fe/2S centers of the enzyme The of was at does not in either its oxidized or with a and with a of oxygen can be The method of was turnover was by the in urate at turnover was as the in at of xanthine and NAD or molecular oxygen The enzyme was between and The enzyme was for the fraction of enzyme was as the fraction of a on of μm xanthine to that on of with NAD at and μm The and the changes are as XDH or XO was with of NAD at The was in a and was at for The of was as the of NAD that the enzyme that no NAD was by the NAD was by its NAD was by isothermal titration as by T. Biochem. 1989; PubMed Scopus Google Scholar) enzyme in the by a XO and XDH prepared by the method of (6Komai H. Massey V. Palmer G. J. Biol. Chem. 1969; 244: 1692-1700Abstract Full Text PDF PubMed Google Scholar). The as as that than of the FAD been XO with and 1-deaza-FAD with flavin The of and 1-deaza-FAD to XDH in binding, as The of is not significantly on binding to XO or The at of is Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of 8-CN-XO that of XO has an of that only of the 8-CN-XO The at of binding to XDH is flavin in the 8-CN-XDH with the flavin was from the enzyme samples in a The of the that of the that the the of 8-CN-XO and 8-CN-XDH are in and The of 1-deaza-XO and 1-deaza-XDH are to that for 1-deaza-XO Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of enzyme the at of 1-deaza-FAD to be for 1-deaza-XO and for are to that of for 1-deaza-XO Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and to that of for the 1-deaza-FAD Schopfer L.M. Massey V. 1990; PubMed Scopus Google Scholar). indicate binding of 1-deaza-FAD to XO and the of enzyme with 1-deaza-FAD that of the 1-deaza-FAD of 1-deaza-XO and 1-deaza-XDH are in A and The redox of the determined to that the FAD potential been in the and Enzyme samples in by by or by an to was between or to the the 2Fe/2S centers as redox and are to be the the higher potential as the 2Fe/2S centers to be of too low a potential to be of as redox from the of μm 8-CN-XO by are in to of the oxidized and the containing the is The at to be the form of Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the is has an to oxidized of Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is from the in A that at the of is no of 2Fe/2S is no in the at than The potential is higher than that of the 2Fe/2S are by changes for are in changes of 2Fe/2S from the two 2Fe/2S centers are 2Fe/2S 2Fe/2S FAD from the two 2Fe/2S centers are FAD FAD are in with the in at which indicate to oxidized changes for 2Fe/2S centers are on of XO (6Komai H. Massey V. Palmer G. J. Biol. Chem. 1969; 244: 1692-1700Abstract Full Text PDF PubMed Google Scholar). changes for oxidized and are from and changes for flavin are from of and from the of and Massey Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and from that of Schopfer L.M. Massey V. 1990; PubMed Scopus Google from the two 2Fe/2S centers are J. Biol. Chem. Full Text PDF PubMed Google in a are in with the in at which indicate to oxidized changes for 2Fe/2S centers are on of XO (6Komai H. Massey V. Palmer G. J. Biol. Chem. 1969; 244: 1692-1700Abstract Full Text PDF PubMed Google Scholar). changes for oxidized and are from and changes for flavin are from of and from the of and Massey Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and from that of Schopfer L.M. Massey V. 1990; PubMed Scopus Google Scholar). from the of μm 8-CN-XO and μm are in The of oxidized and and the of oxidized and as and The for the was the for the flavin of to the of which is by a of as with a of for the of the The a potential of for the of the 8-CN-FAD. a in the potential of of 40 on binding to a in with of and on the binding of native FAD and to XO and of ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± not ± ± ± ± ± ± not in a from the of μm 8-CN-XDH are in The with respect to the of 8-CN-XO is that the is the as by the from to Massey V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is with the of in XDH T. Nishino T. Massey V. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of A versus A from the of a μm of 8-CN-XDH a in with the low of the of of the is to a of a of the the of native FAD J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of the that the and the are by To the potential of the in XDH, the enzyme was an and in the of at and in a in the of at The of oxidized and and the of oxidized and flavin as and The from the of 8-CN-XDH with are in of the of and of the of versus the for the of of and for the a as both and are However, of and obtained. To the potential of the in XDH, the enzyme was an as in the of at The of oxidized and and the of oxidized and flavin as and A of the of versus the of the a of for the is in to a of an potential of and of the The in is to a as is a A of was obtained. the of the are not the are in A of the potential of from to on binding to XDH is in with with other of NAD with native xanthine oxidase and xanthine ± ± ± ± ± ± ± ± ± ± as and at μm xanthine and μm oxygen for XO and at μm μm and no oxygen for Enzyme was for by to at V. Palmer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). are as xanthine/oxygen rate for XO and as xanthine/NAD rate for in a as and at μm xanthine and μm oxygen for XO and at μm μm and no oxygen for Enzyme was for by to at V. Palmer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). are as xanthine/oxygen rate for XO and as xanthine/NAD rate for from a of μm 1-deaza-XO are in is only of the which is as an the of 1-deaza-FAD has M. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of 2Fe/2S by the of a low potential for is in the the flavin to have a potential than the 2Fe/2S is with the of and Massey at Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to the in at and The midpoint potential determined at of is to that of determined at Massey V. J. Biol. Chem. Full Text PDF PubMed Google the potential of be for a of The potential of is similar to that of native XDH, J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that with 1-deaza-FAD does a low flavin potential form of XO. The of μm 1-deaza-XDH is in for significantly flavin 1-deaza-XDH to The of the two are similar to each other The redox determined from are and The midpoint potential of is than that of native The of the to xanthine/oxygen and xanthine/NAD turnover was to the of the flavin midpoint potential in oxygen of both and oxidizing substrates of with a from are in The of xanthine/oxygen is to that of native C.M. Massey V. K. and Google Scholar). that the flavin is and the of to the of the flavin midpoint potential on Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The high with the oxygen for turnover as as the xanthine/oxygen turnover of 8-CN-XDH is of the only than the of oxygen at of the XO forms catalyzes a of xanthine/NAD be of a of dehydrogenase-type the of the are of The 8-CN-XDH is in xanthine/oxygen of However, no of xanthine/NAD turnover was The on of 8-CN-XDH is evidence that the is a dehydrogenase-type of the to was on 8-CN-XDH with μm the similar with native XO in no of was at that 8-CN-XDH is a dehydrogenase-type the of xanthine/NAD activity. The xanthine/NAD of 8-CN-XDH that a low flavin midpoint potential is for xanthine/NAD turnover of 1-deaza-XO has of similar to that at Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The is a enzyme The xanthine/NAD of of that the low flavin midpoint potential of is not sufficient to dehydrogenase activity. of 1-deaza-XDH is that of native XDH, that with flavin has not the The xanthine/NAD of 1-deaza-XDH is not a Nishino T. Nishino T. Schopfer L.M. Massey V. J. Biol. Chem. 1989; 264: 6075-6085Abstract Full Text PDF PubMed Google Scholar) that of XDH with the low potential and in enzyme containing xanthine/NAD of the native enzyme at a of T. Nishino T. Schopfer L.M. Massey V. J. Biol. Chem. 1989; 264: 6075-6085Abstract Full Text PDF PubMed Google Scholar). The low potential of 1-deaza-XDH in a fraction of flavin To the of substrate binding and the for the flavin midpoint potential to be or that of its electron the of native XO and XDH to a of of different was was at μm oxygen, for xanthine/NAD is a higher of xanthine/oxygen than that in for for by to the at V. Palmer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the potential of the electron acceptor as high as than the flavin potential of XO Palmer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar), has no on the of XO. the from the 1-deaza-XO that thermodynamics are not sufficient for dehydrogenase activity. the potential of the acceptor only on the of is not as by xanthine is to be the in J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The flavin of oxidized XDH is to be on the of the NAD analog J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google and T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google and the in to the changes are on the of to μm XDH, NAD has a on the of XDH T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). No changes on the of either to oxidized XO that NAD does not to XO to the NAD binding by was to distinguish XO has a or is a binding and is not in the of the The be that of NAD binding to the d of NAD binding to XDH of μm has been C.M. Massey V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The of NAD to XO in the with oxidation of a fraction of XO and not with any XO and NAD no binding, binding of NAD to the oxidized was μm of oxidized XDH and XO each with of NAD from to of the NAD was from the of the the was in a d of 280 ± 145 μm was for the binding of NAD to XDH, but no evidence could be binding of NAD to XO. NAD binding to XDH was by a different that of isothermal titration T. Biochem. 1989; PubMed Scopus Google Scholar), a d of 160 ± 40 μm not in a no of NAD binding to the oxidase form could be obtained. The of the was to the of the low flavin midpoint potential and of NAD binding in oxidizing substrates for XO and The flavin midpoint potential was by enzyme samples with FAD redox of enzyme forms that the changes in potential of 8-CN-XDH in an enzyme with a flavin midpoint potential of to be too high to efficiently reduce at Scholar). enzyme was in xanthine/NAD was A low flavin potential is necessary for NAD of 1-deaza-XO an oxidase-type enzyme with a flavin midpoint potential to that of native as as an 1-deaza-XO has no dehydrogenase than does native XO. could be of a of dehydrogenase-type the of native XO and XDH in NAD of higher redox that only dehydrogenase-type enzyme could as an oxidizing indicate that thermodynamics are but not for dehydrogenase activity. NAD binding is an The of XO is not the of NAD or of that of XDH T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). NAD does not xanthine/oxygen turnover of XO. μm μm oxygen, and μm xanthine/oxygen turnover at the rate in the absence of NAD xanthine/oxygen turnover be in of xanthine/NAD turnover (9Harris C.M. Massey V. J. Biol. Chem. 1997; 272: 8370-8379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The of an NAD binding in XDH, but not in was by the of NAD in of NAD and XO or XDH and by isothermal titration calorimetry. d for NAD binding to oxidized XDH 280 ± 145 μm and 160 ± 40 binding of NAD to XDH is with a d Nishino and Nishino a d of μm for NAD binding to oxidized XDH T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). NAD binding to the of XDH has been to be the of NAD binding to oxidized enzyme forms C.M. Massey V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). binding with the of on the of xanthine/NAD of and the of xanthine/oxygen to the of NAD indicate that the changes that XDH to XO the in XO. The binding are with the oxidized NAD binding to XO and XDH is not to for XO. that oxygen binding is not an in substrate XDH has been to react with oxygen by (9Harris C.M. Massey V. J. Biol. Chem. 1997; 272: 8370-8379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and is no evidence for oxygen binding to XO or any other oxidase V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). NAD binding to oxidized XDH, d is than to d of μm J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). d and as the flavin midpoint potential of XDH J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a flavin midpoint potential of can be for XDH on a in flavin potential of be at the of electron at the flavin and at the and molybdenum in oxidation with NAD C.M. Massey V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and in with J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar), electron into and of the flavin to only in the absence of electron to is only NAD from the with NAD C.M. Massey V. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) indicate that of NAD the of substrate binding is to the midpoint potential of the the between FAD and NAD from to The d of μm for NAD binding to oxidized XDH T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar) is higher than that for the μm L.M. Massey V. Nishino T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). samples of XO with a of midpoint and Massey Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar) a between in and the fraction of flavin in the the samples of native XDH, and 1-deaza-XO in the of with respect to their xanthine/oxygen both 8-CN-XO and 8-CN-XDH have of 8-CN-XO is to whereas at 8-CN-XDH that the of and Massey was at a higher a flavin potential for NAD J. Massey V. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Nishino T. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J. Massey V. J. Biol. Chem. Full Text PDF PubMed Google L.M. Massey V. Nishino T. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and preference for NAD oxygen as a substrate (9Harris C.M. Massey V. J. Biol. Chem. 1997; 272: 8370-8379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), native XDH a low potential oxidase-type enzyme with respect to its xanthine/oxygen turnover the absence of a number of and oxidase the of a that the XO form have to for the of and Y. V. for of for of XDH and and for the isothermal
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