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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 greater than or equal to 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.
Highlights
Differences in Protein Structuroef Xanthine Dehydrogenaseand Xanthine Oxidase Revealed by Reconstitution with Flavin Active Site Probes*
The deflavoenzymes, attention to the similarities and differences found between stillretainingtheir molybdopterin andiron-sulfur the chicken liver xanthine dehydrogenase and the much studprosthetic groups, were reconstituted with a series of ied milk xanthine oxidase(see Bray, 1975 and Coughlan, FAD derivatives containingchemically reactive or en- 1980, forextensive reviews ontheproperties of theseenvironmentally sensitive substituents in the isoalloxazine ring system
In an attempt to define better the structural differences between the two proteins, which despite having similar molecular weights and identical cofactor composition have freely accessible to solvent in milk xanthine oxi- very different patternsof reactivity with pyridinenucleotides dase, but was significantly less exposed to solvent in and molecular oxygen, we decided to employ the strategy of the chicken liver dehydrogenase
Summary
Milk xanthine oxidase and chicken liver xanthine dehydro- of a negativelycharged region in the proteinw,hich prevented genase share many properties Thedehydrogenase form, with intact NADbinding site, and a strong negative charge located near the flavin N(1)-position, would change its conformation on oxidation of the crucial thiol residues in such a manner that the NAD-binding site was lost at thesame time asthe negatively charged protein residue was removed from the vicinity of the flavin N(l)-position, andreplaced instead by a partialpositive charge, such ascould be provided from amide nitrogens or the N-terminal region of an a-helix Evidence that such conformational changes may occur in a reversible fashion with rat liver xanthine dehydrogenase has been obtained in a similar study using the same flavin derivatives as reported here, and will be described in full in a future p~blication.~. We are indebted to Dr Ken Fish, University of Michigan, for the gift of milk xanthine oxidase
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