https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(02)00003-8.pdf

Structure, mechanism and regulation of peroxiredoxins

The thioredoxin antioxidant system.

Short protocols in molecular biology

The OxyR transcription factor is sensitive to oxidation and activates the expression of antioxidant genes in response to hydrogen peroxide in Escherichia coli. Genetic and biochemical studies revealed that OxyR is activated through the formation of a disulfide bond and is deactivated by enzymatic reduction with glutaredoxin 1 (Grx1). The gene encoding Grx1 is regulated by OxyR, thus providing a mechanism for autoregulation. The redox potential of OxyR was determined to be -185 millivolts, ensuring that OxyR is reduced in the absence of stress. These results represent an example of redox signaling through disulfide bond formation and reduction.

https://science.sciencemag.org/content/sci/279/5357/1718.full.pdf

Activation of the OxyR Transcription Factor by Reversible Disulfide Bond Formation

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as ‘damage’ or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid1,2. Cysteine–sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine–sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass Mr = 13,000, which we have named sulphiredoxin (identified by the US spelling ‘sulfiredoxin’, in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine–sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides3,4,5 and control hydroperoxide-mediated signalling in mammals6,7,8. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine–sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine–sulphinic acid modifications, and in signalling pathways involving protein oxidation.

ATP-dependent reduction of cysteine–sulphinic acid by S. cerevisiae sulphiredoxin

The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide-oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative containing one additional oxygen relative to the Cys-S-NBD product. The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-Cl over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.

Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase.

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.

/pdf/roles-for-the-two-cysteine-residues-of-ahpc-in-catalysis-of-1ol6bkh6jl.pdf

Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties. Recombinant proteins were isolated in high yield (25−33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. coli cells. We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein. All assays gave identical results whether overexpressed S. typhimurium proteins from E. coli or proteins isolated directly from S. typhimurium were used. Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system. AhpF possesses multiple pyridine nuc...

Flavin-Dependent Alkyl Hydroperoxide Reductase from Salmonella typhimurium. 1. Purification and Enzymatic Activities of Overexpressed AhpF and AhpC Proteins†

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

C165S AhpC in its sulfenate (Cys-SO-) and presumed thiolate (Cys-S-) forms at pH 7 (pKa for sulfenic acid about pH 6.1) exhibit low extinction absorbance bands around 367 and 324 nm, respectively. Sulfenic acid content of the protein can be assessed by its reactivity with the chromophoric TNB anion. Using this technique, H2O2 titrations of C165S AhpC give a maximum of about 1 SOH per subunit on addition of 1.0 to 1.2 equivalents of H2O2. Cys46-SO- is moderately air stable at neutral pH and room temperature and is oxidized at a steady rate of about 10% per half hour. Cys46-SO- of C165S AhpC is reduced in the presence of catalytic amounts of AhpF by approximately 1 equivalent of NADH to regenerate the Cys46-S- species. NBD chloride is extremely useful as a trapping agent for cysteine sulfenic acid. The Cys46-S(O)-NBD adduct absorbs maximally at 347 nm and is 16 amu larger than the Cys46-S-NBD adduct (lambda max = 420 nm) as shown by ESI-MS. Other electrophilic thiol reagents also react with Cys46-SO-; however, iodoacetamide and N-ethylmaleimide reactivities are much lower with Cys46-SO- than with Cys46-S-. These methods are applicable to other sulfenic acid-containing proteins, although in some cases the proteins must be denatured in order to provide accessibility of this species toward labeling agents.

Holly R. Ellis

Papers

Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase.

Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

Flavin-Dependent Alkyl Hydroperoxide Reductase from Salmonella typhimurium. 1. Purification and Enzymatic Activities of Overexpressed AhpF and AhpC Proteins†

AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase.

Identification of cysteine sulfenic acid in AhpC of alkyl hydroperoxide reductase.