PURIFICATION AND CHARACTERIZATION OF A FLAVOPROTEIN INVOLVED IN THE DEGRADATION OF EPOXYALKANES BY XANTHOBACTER PY2

Recently a newly discovered pyridine nucleotide-disulfide oxidoreducta se was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bent, J. A. M., Westphal. A. & De Kok, A. (1996) J. Bacteriol. 178, 6644-6646]. The disulfide oxidore ductase has now been purified front propene-grown Xanthobacter Py2. Th is enzyme (component II) is a NADPH-dependent FAD-containing homodimer ic protein, The physiological substrate for this enzyme is unknown. Th e enzyme was active with the following dithiol substrates in decreasin g order: 1,3-propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5.5'-dithiobis(2-nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5 .5'-dithiobis(2-nitrobenzoate) and a low diaphorase and oxidase activi ty, Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol sho w that dir enzyme operates by a ternary complex mechanism in the direc tion of NADP(+) reduction. Anaerobic incubation of the enzyme with 1,3 -propanedithiol resulted in slow reduction of the enzyme to yield the thiolate-FAD charge-transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP(+) with the enzyme-propanedithiol mixture resulted in the formati on of a complex between the reduced enzyme and NADP(+) within the dead time of the instrument (5.6 ins). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)-thiol ad duct, as judged from the spectral changes. This suggests that the rate -limiting step is the transfer of a hydride ion from the half-reduced enzyme to NADP(+). Stopped-flow experiments involving reduction by NAD PH show a biphasic behavior. The rapid formation (k(obs) = 40 s(-1)) o f a transient intermediate with little absorption decrease at 460 nm a nd long wavelength absorption was followed by the slow formation (k(ob s) = 4 s(-1)) of a species characterized as the thiolate-FAD charge-tr ansfer complex with bound NADP(+). Some formation of the FAD C(4a)-thi ol adduct was also observed. Photoreduction in the presence of deazafl avin results in rapid bleaching at 450 nm. followed by the slow format ion of a stable semiquinone. Full reduction could not be achieved, tit her by photoreduction or with NADPH, and was incomplete even with dith ionite or NADPH in the presence of arsenite. The results indicate a lo w odor potential of the FAD and a slow rate of electron transfer from the pyridine nucleotide to the redox active disulfide and vice versa, From a sequence alignment with other disulfide reductases, it appears that the active site His Glu diad is absent in this enzyme. The kineti c and spectral features described above will be discussed in this cont ext.