Abstract
Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds.
Highlights
15020 JOURNAL OF BIOLOGICAL CHEMISTRY change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds
Trx is a ubiquitous oxidoreductase whose catalytic activity depends on a conserved CXXC motif that is maintained reduced by thioredoxin reductase at the expense of NADPH, the best characterized function of Trx is the reduction of disulfide bonds that form in proteins during catalysis or as a result of oxidative stress [7], and Trx has recently been shown to catalyze the direct denitrosylation of proteins in vivo [1]
If DsbC and DsbG are able to reduce GSNO, this should lead to the oxidation of their catalytic CXXC motifs, which can be monitored using AMS trapping experiments
Summary
DsbG and DsbC Are Poor Denitrosylating Enzymes—As a first step, we asked whether DsbC and DsbG are able, like Trx-1, to catalyze the in vitro reduction of GSNO. Two hydrogen bonds to the nucleophilic cysteine are lost (using a cutoff of 3.5 Å for a hydrogen bond) (Fig. 6C and Table 1): one between the backbone oxygen of Met200 and the sulfur of nucleophilic Cys109 and one caused by absence of the side chain of the Thr. In the oxidized and reduced form of wild type DsbG, the main chain O and O␥ of Thr200 are within H-bond distance with the S␥ of Cys109 (Fig. 6), which has been shown to destabilize the oxidized form of the protein [14, 17]. In the oxidized and reduced form of wild type DsbG, the main chain O and O␥ of Thr200 are within H-bond distance with the S␥ of Cys109 (Fig. 6), which has been shown to destabilize the oxidized form of the protein [14, 17] The lack of this interaction from Met200 in the DsbGCGPC-T200M mutant could lead to the observed stabilization of the oxidized form. Whereas DsbGCGPC is found in the reduced form in both chains in the asymmetric
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