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 disulfide bond formation in vivo, uses the oxidizing power of quinones to generate disulfides de novo. This is a novel catalytic activity, which to our knowledge has not yet been described. This catalytic activity is apparently the major source of disulfides in vivo. We developed a new assay to characterize further this previously undescribed enzymatic activity, and we show that quinones get reduced during the course of the reaction. DsbB contains a single high affinity quinone-binding site. We reconstitute oxidative folding in vitro in the presence of the following components that are necessary in vivo: DsbA, DsbB, and quinone. We show that the oxidative refolding of ribonuclease A is catalyzed by this system in a quinone-dependent manner. The disulfide isomerase DsbC is required to regain ribonuclease activity suggesting that the DsbA-DsbB system introduces at least some non-native disulfide bonds. We show that the oxidative and isomerase systems are kinetically isolated in vitro. This helps explain how the cell avoids oxidative inactivation of the disulfide isomerization pathway.
Bader
M W
MW
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
Xie
T
T
Yu
C A
CA
Bardwell
J C
JC
D001426:Bacterial Proteins / Q000378:metabolism*; D016227:Benzoquinones / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014451:Ubiquinone / Q000378:metabolism
D001426:Bacterial Proteins; D016227:Benzoquinones; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D014451:Ubiquinone; C004532:quinone; D010088:Oxidoreductases; D012259:Ribonuclease, Pancreatic; D019704:Protein Disulfide-Isomerases