Purification and characterization of a flavoprotein involved in the degradation of epoxyalkanes by Xanthobacter Py2

Recently a newly discovered pyridine nucleotide-disulfide oxidoreductase 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 oxidoreductase has now been purified front propene-grown Xanthobacter Py2. This enzyme (component II) is a NADPH-dependent FAD-containing homodimeric protein, The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing 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 activity, Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol show that dir enzyme operates by a ternary complex mechanism in the direction 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 formation 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 adduct, 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 NADPH show a biphasic behavior. The rapid formation (k(obs) = 40 s(-1)) of a transient intermediate with little absorption decrease at 460 nm and long wavelength absorption was followed by the slow formation (k(obs) = 4 s(-1)) of a species characterized as the thiolate-FAD charge-transfer complex with bound NADP(+). Some formation of the FAD C(4a)-thiol adduct was also observed. Photoreduction in the presence of deazaflavin results in rapid bleaching at 450 nm. followed by the slow formation of a stable semiquinone. Full reduction could not be achieved, tither by photoreduction or with NADPH, and was incomplete even with dithionite or NADPH in the presence of arsenite. The results indicate a low 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 kinetic and spectral features described above will be discussed in this context.