Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase cat alyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia col i, have demonstrated that the purified protein utilizes NADPH as a facile e lectron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. BioE. Chem. 2 72, 3355-3362] that, at pH 8 and in the presence of saturating concentratio ns of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app)=5 00 s(-1) K-m,K-app = 269 muM, and k(cat,app)/K-m,K-app = 1.86 x 10(6) M-1 s (-1)) compared to NADH (k(cat),(app) 47 s(-1) K-m,K-app = 394 muM, and K-ca t,K-app/K-m,K-app 1.19 x 10(5) M-1 s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzy me also utilized ferricyanide as an artificial electron acceptor, which eff ectively suppressed biotin sulfoxide reduction and biotin formation. Analys is of the enzyme isolated from tungsten-grown cells yielded decreased reduc ed methyl viologen:BSO reductase, NADPH: BSO reductase, and NADPH:FR activi ties, confirming that Mo is required for all activities. Kinetic analyses o f substrate inhibition profiles revealed that the enzyme followed a Ping Po ng Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive su bstrate inhibition. Replots of the 1/nu -axes intercepts of the parallel as ymptotes obtained at several low concentrations of fixed substrate yielded a K-m for BSO of 714 and 65 muM for NADPH. In contrast, utilizing NADH as a n electron donor, the replots yielded a K-m for BSO of 132 muM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yiel ded a Ki for BSO of 6.10 mM and 900 muM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibit ed NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpy rocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histi dine residues are involved in cofactorbinding.