Disulfide‐bond formation in the H⁺‐pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Abstract

Redox control of disulfide-bond formation in the H + -pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K + -independent (type II) H + -pyrophosphatases.

Mechanism of Cd and Cu action on the tonoplast proton pumps in cucumber roots

“…Such results suggested the existence of in vivo redox control of plant V‐ATPases, as it was earlier evidenced for the yeast enzyme (Oluwatosin and Kane ). Redox‐dependent modulation of the activity as a consequence of disulfide bond formation has also been postulated as a mechanism of regulation of H + ‐PPases (Mimura et al ). The authors pointed out the possibility of cooperative inactivation and reactivation of vacuolar PPase and ATPase under oxidative and reductive conditions.…”

“…A similar mechanism has been proposed for the regulation of H + ‐PPases. The activity of Streptomyces coelicolor enzyme expressed in Escherichia coli was affected by its redox state as a consequence of disulfide bond formation between Cys253 and Cys621 (Mimura et al ). A second biochemical modification controlling proton‐pump activity is reversible phosphorylation and interaction with 14‐3‐3 protein.…”

Plant proton pumps

“…Site‐directed thiol cross‐linking and selective oxidation experiments with the H + ‐PPase of ScPP suggest that the higher order oligomeric state of this enzyme consists of at least two or three sets of dimers [90] and that two opposing cysteine residues (Cys 253 and Cys 621 ) are required for reversible enzyme inactivation by oxidation [91]. As all known type II, but not type I, H + ‐PPases share cysteine residues corresponding to the Cys 253 and Cys 621 of ScPP, the authors speculate that redox control of enzyme activity could be unique to type II H + ‐PPases [91]. However, as Cys 621 of ScPP is conserved among type I H + ‐PPases, the authors further speculate that intermolecular disulfite cross‐linking might be a redox mechanism for the regulation of type I H + ‐PPases [91].…”

“…As all known type II, but not type I, H + ‐PPases share cysteine residues corresponding to the Cys 253 and Cys 621 of ScPP, the authors speculate that redox control of enzyme activity could be unique to type II H + ‐PPases [91]. However, as Cys 621 of ScPP is conserved among type I H + ‐PPases, the authors further speculate that intermolecular disulfite cross‐linking might be a redox mechanism for the regulation of type I H + ‐PPases [91]. These findings suggest that plant H + ‐PPases may be regulated in a manner similar to what is seen in the barley V‐ATPase which has been reported to be inhibited by H 2 O 2 and reactivated by reduced glutathione in vitro [92].…”

Zinc‐binding and structural properties of the histidine‐rich loop of Arabidopsis thaliana vacuolar membrane zinc transporter MTP1

“…S2. The dimer formation is due to a disulfide bond between the monomers, because dimer formation was stimulated by Cu(II)‐(1,10‐phenanthroline) 3 , which is an oxidizing agent that stimulates the formation of disulfide bonds [21], and was cleaved by dithiothreitol (Fig. S3).…”