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 nucleotide-dependent activities [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhydrogenase, and, in the presence of AhpC, peroxide reductase activities]. Although AhpF can use either NADH or NADPH as the electron donor for these activities, NADH is the preferred reductant (Km,app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADPH when analyzed using DTNB reductase assays). Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities. In contrast, thiol modification of reduced AhpF does not affect transhydrogenase or oxidase activities. These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides within each protein.

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.

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 ROleaving 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.The alkyl hydroperoxide reductase system from Salmonella typhimurium protects the cell from the toxic effects of reactive oxygen species generated during normal aerobic metabolism and during times of oxidative stress (1-3). The system consists of two proteins, AhpF and AhpC, that together catalyze pyridine nucleotide-dependent reduction of organic hydroperoxides and H 2 O 2 (2, 4-6). The genes encoding these two proteins are located on the same operon and are under the transcriptional control of the global stress regulator OxyR (3, 7). Our previous studies have demonstrated that the 57 kDa AhpF is a thioredoxin reductase-like pyridine nucleotide-dependent protein that contains two redox-active disulfide centers and one FAD per subunit (4-6). The 21 kDa AhpC protein contains two redox-active cystine disulfide bonds per dimer that have been implicated in the reduction of organic hyd...