KINETIC ISOTOPE EFFECTS AND ELECTRON-TRANSFER IN THE REDUCTION OF XANTHINE OXIDOREDUCTASE WITH 4-HYDROXYPYRIMIDINE - A COMPARISON BETWEEN OXIDASE AND DEHYDROGENASE FORMS

Isolated from bovine milk, xanthine oxidase (XO) and xanthine dehydrog enase (XDH) are two interconvertible forms of the same protein, differ ing in the number of protein cysteines versus cystines. Most differenc es between XO and XDH are localized to the FAD center, the site at whi ch the oxidizing substrates NAD and molecular oxygen react. A comparat ive study of the reduction of XO and XDH has been performed to assess differences in reactivity of the molybdopterin site, as well as subseq uent electron-transfer events from molybdenum to 2Fe/2S and FAD center s. The compound 4-hydroxypyrimidine (4-OH-P) was chosen as reducing su bstrate because its higher K-m value raised the possibility of binding weak enough to measure kinetically, and its high k(cat) value could a llow detection of intramolecular electron-transfer reactions. As measu red by stopped flow spectrophotometry, XO and XDH react with the first equivalent of 4-OH-P via similar mechanisms, differing in the magnitu de of rate and dissociation constants. Using [2-H-2]4-OH-P as substrat e, a (D)(k/K-d,) isotope effect of 1.9 to 2.3 suggests that movement o f the hydrogen abstracted from substrate appreciably limits the rate o f initial enzyme reduction from Mo(VI) to Mo(IV). Monitoring the visib le spectrum of the enzymes, the first observed step is reduction of a single 2Fe/2S center and presumably re-oxidation of Mo(IV) to Mo(V). T his suggests a common pathway for electron transfer involving reductio n of a 2Fe/2S center prior to reduction of the second 2Fe/2S and FAD c enters. Rates of the first electron transfer from molybdenum to the 2F e/2S center are rapid, 290 s(-1) with XO and 180 s(-1) with XDH, and a re consistent with rates measured by flash photolysis (Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 3 0, 5912-5917) allowing discrete observation of the electron-transfer r eactions that occur during turnover. This step also exhibits a modest primary kinetic isotope effect of 1.5 to 1.6 when [2-H-2]4-OH-P is use d, possibly due to deprotonation of the molybdenum center prior to ele ctron transfer. A second one-electron transfer, presumably oxidizing M o(V) to Mo(VI), follows in a step coincident with product dissociation , consistent with a role for product release in controlling electron t ransfer events. The kinetics of this complex system are described and interpreted quantitatively in models that are consistent with all the data.