Disulfide bonds are generated by quinone reduction

The chemistry of disulfide exchange in biological systems is well studied. However, very little information is available concerning the actual origin of disulfide bonds. Here we show that DsbB, a protein required for disulfid e bond formation in vivo, uses the oxidizing power of quinones to generate disulfides de novo, This is a novel catalytic activity, which to our knowle dge has not yet been described. This catalytic activity is apparently the m ajor source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that q uinones get reduced during the course of the reaction. DsbB contains a sing le high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vi vo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonu clease A is catalyzed by this system in a quinone-dependent manner. The dis ulfide isomerase DsbC is required to regain ribonuclease activity suggestin g that the DsbA-DsbB system introduces at least some non-native disulfide b onds. We show that the oxidative and isomerase systems are kinetically isol ated in vitro. This helps explain how the cell avoids oxidative inactivatio n of the disulfide isomerization pathway.