Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes

V. Massey, L. M. Schopfer, T. Nishino, T. Nishino

Research output: Contribution to journalArticle

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Abstract

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent ≥4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.

Original languageEnglish (US)
Pages (from-to)10567-10573
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number18
StatePublished - Jan 1 1989

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Xanthine Dehydrogenase
Xanthine Oxidase
Catalytic Domain
Oxidoreductases
Flavins
Liver
Flavin-Adenine Dinucleotide
Chickens
Milk
Proteins
4,6-dinitro-o-cresol
Enzymes
Prosthetics
Sulfur
Iron
Binding Sites
Derivatives

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. / Massey, V.; Schopfer, L. M.; Nishino, T.; Nishino, T.

In: Journal of Biological Chemistry, Vol. 264, No. 18, 01.01.1989, p. 10567-10573.

Research output: Contribution to journalArticle

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