Role and oxidation state of the pterin molybdenum cofactor of molybdenum enzymes: studies of a Drosophila melanogaster xanthine dehydrogenase (rosy) variant, G1011E.

Xanthine dehydrogenase is one of the family of enzymes known as molybdenum-containing hydroxylases [I] . These are complex enzymes, containing two [2Fe-2S] clusters, FAD and the pterin molybdenum cofactor [2]. In Drosophrlu nzelutioguster, xanthine dehydrogenase is encoded by the rosy gene, expression of which affects the eye colour of the flies [3]. More than 200 rosy mutant strains are available, of which at least 23 are known [4] to involve specific amino acid changes in the protein sequence. We report preliminary molecular characterisation of the xanthine dehydrogenase variant from one of these strains, 91544, containing the molybdenum domain [ I ] mutation GI01 IE. Preliminary studies of the G I0 I 1 E enzyme using nine different assays showed an activity pattern quite unlike that of the wild-type enzyme. While full activity was retained towards NADH as reducing substrate, only one activity that involved a reducing substrate reacting at the molybdenum site was observable, xanthine: PMS/cytochrome-c. An explanation of this unusual pattern was provided by detailed study of the behaviour of the purified enzyme in the xanthine: PMSicytochrome-c assay. With the mutant enzyme, in contrast to the wild-type, maximal activity was not exhibited immediately on the addition of xanthine but developed progressively over a period of several minutes (Fig.1). Extrapolation indicated zero activity at zero-time, suggesting that the GI 0 I I E enzyme became active only after modification, possibly by oxidation, by either PMS or cytochrome-c or both. It was postulated that this activation might generate in the GI01 IE enzyme other activities that it initially lacked, e.g. xanthine: NAD+. A series of experiments was performed in which the GlOl lE enzyme was pre-incubated with several different reagents. After treatment the enzyme was assayed for both xanthine: NAD' and xanthine: PMS/cytochrome-c activites (Table 1). In the latter assay both maximal and extrapolated zero-time activities were calculated. Activation is therefore shown by either the appearance of activity in the xanthine: NAD' assay or the presence of activity at zero-time in the xanthine: PMS/cytochrome-c assay. These studies confirmed that mild oxidation, brought about by either femcyanide or a