Coenzyme A-disulfide reductase from Staphylococcus aureus: Evidence for asymmetric behavior on interaction with pyridine nucleotides

unusual flavoprotein disulfide reductase, which catalyzes the NADPH-depende nt reduction of CoASSCoA, has recently been purified from the human pathoge n Staphylococcus aureus [delCardayre, S. B., Stock, K. P., Newton, G. L., F ahey, R. C., and Davies, J. E. (1998) J. Biol. Chem. 273, 5744-5751], Coenz yme A-disulfide reductase (CoADR) lacks the redox-active protein disulfide characteristic of the disulfide reductases; instead, NADPH reduction yields I protein-SH and 1 CoASH, Furthermore, the CoADR sequence reveals the pres ence of a single putative active-site Cys (Cys43) within an SFXXC motif als o seen in the Enterococcus faecalis NADH oxidase and NADH peroxidase, which use a single redox-active cysteine-sulfenic acid in catalysis. In this rep ort, we provide a detailed examination of the equilibrium properties of bot h wild-type and C43S CoADRs, focusing on the role of Cys43 in the catalytic redox cycle, the behavior of both enzyme forms on reduction with dithionit e and NADPH, and the interaction of NADP(+) with the corresponding reduced enzyme species. The results of these analyses, combined with electrospray m ass spectrometric data for the two oxidized enzyme forms, fully support the catalytic redox role proposed for Cys43 and confirm that this is the attac hment site for bound CoASH. In addition, we provide evidence indicating dra matic thermodynamic inequivalence between the two active sites per dimer, s imilar to that documented for the related enzymes mercuric reductase and NA DH oxidase; only I FAD is reduced with NADPH in wild-type CoADR. The EH2. N ADPH/EH4.NADP(+) complex which results is reoxidized quantitatively in titr ations with CoASSCoA, supporting a possible role for the asymmetric reduced dimer in catalysis.