5.A.2 The Disulfide Bond Oxidoreductase-B (DsbB) Family
In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD (Ito and Inaba, 2008). In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domains and other regulatory domains lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one.
The proteins of the DsbB family transfer electrons across the bacterial cytoplasmic membrane from periplasmic dithiol proteins to quinones in the membrane. The reduced quinones then reduce cytoplasmic electron acceptors such as O2via cytochrome oxidases (cytochrome d and cytochrome o, respectively, in E. coli) as well as nitrate via nitrate reductase and fumarate via fumarate reductase.
The pathway for electron transfer via DsbB is:
reduced proteinperiplasm → DsbAperiplasm → DsbBmembrane → quinonesmembrane →
reductasemembrane → terminal electron acceptorcytoplasm
DsbB proteins and their homologues are found only in Gram-negative bacteria. The proteins are small (163-266 aas) and exhibit 4 TMSs. The E. coli protein has 4 essential cysteine residues, reversibly forming two disulfide bonds, both on the periplasmic side of the membrane. They occur at position C41/C44 and C104/C130 (Kobayashi et al., 2001). An essential arginyl residue, R48 (Kadokura et al., 2000), is fully conserved in the DsbB homologues. It has been reported that DsbB can function in both electron donation and acceptance with the periplasmic thioredoxin I (Debarbieux and Beckwith, 2000). However, electron transfer to a terminal electron acceptor such as O2 renders the process essentially irreversible. The mechanism of electron transfer involves intramolecular disulfide exchange. Only fully reduced C41-C44 can be reoxidized by ubiquinone (Grauschopf et al., 2003).
In Rhodobacter capsulatus, DsbB, acts as an 'electron conduit' between the hydrophilic metalloid, tellurite (TeO3-2 and the lipid-embedded Q pool. Thus, in habitats contaminated with subinhibitory amounts of TeIV, the metalloid is likely to function for disposal of the excess reducing power in facultative phototrophic bacteria (Borsetti et al., 2007).
The crystal structure of the DsbB-DsbA complex at 3.7 Å resolution reveals four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. Thus, DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.
Zhou et al., 2008 described the NMR structure of DsbB, which reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. The structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis, revealed functionally relevant changes induced by these substrates. Dynamics measurements and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated.
Disulfide bond formation is a catalyzed reaction essential for the folding and stability of proteins in the secretory pathway. In prokaryotes, disulfide bonds are generated by DsbB or VKOR homologs that couple the oxidation of a cysteine pair to quinone reduction. Vertebrate VKOR and VKOR-like enzymes have gained the epoxide reductase activity to support blood coagulation. The core structures of DsbB and VKOR variants share the architecture of a four-transmembrane-helix bundle that supports the coupled redox reaction and a flexible region containing another cysteine pair for electron transfer. Despite considerable similarities, recent high-resolution crystal structures of DsbB and VKOR variants reveal significant differences (Li 2023). DsbB activates the cysteine thiolate by a catalytic triad of polar residues, a reminiscent of classical cysteine/serine proteases. In contrast, bacterial VKOR homologs create a hydrophobic pocket to activate the cysteine thiolate. Vertebrate VKOR and VKOR-like proteins maintain this hydrophobic pocket and further evolved two strong hydrogen bonds to stabilize the reaction intermediates and increase the quinone redox potential. These hydrogen bonds are critical to overcome the higher energy barrier required for epoxide reduction. The electron transfer process of DsbB and VKOR variants uses slow and fast pathways, but their relative contribution may be different in prokaryotic and eukaryotic cells. The quinone is a tightly bound cofactor in DsbB and bacterial VKOR homologs, whereas vertebrate VKOR variants use transient substrate binding to trigger the electron transfer in the slow pathway. Overall, the catalytic mechanisms of DsbB and VKOR variants have fundamental differences (Li 2023).
The generalized vectorial electron transfer reaction catalyzed by DsbB is:
2 e- cytoplasm → 2 e- periplasm