Substrate specificity and redox potential of AhpC, a bacterial peroxiredoxin

Abstract

Typical 2-Cys peroxiredoxins (Prxs) are ubiquitous peroxidases that are involved in peroxide scavenging and/or the regulation of peroxide signaling in eukaryotes. Despite their prevalence, very few Prxs have been reliably characterized in terms of their substrate specificity profile and redox potential even though these values are important for gaining insight into physiological function. Here, we present such studies focusing on Salmonella typhimurium alkyl hydroperoxide reductase C component (StAhpC), an enzyme that has proven to be an excellent prototype of this largest and most widespread class of Prxs that includes mammalian Prx I-Prx IV. The catalytic efficiencies of StAhpC (k(cat)/K(m)) are >10(7) M(-1).s(-1) for inorganic and primary hydroperoxide substrates and approximately 100-fold less for tertiary hydroperoxides, with the difference being exclusively caused by changes in K(m). The oxidative inactivation of AhpC through reaction with a second molecule of peroxide shows parallel substrate specificity. The midpoint reduction potential of StAhpC is determined to be -178 +/- 0.4 mV, a value much higher than most other thiol-based redox proteins. The relevance of these results for our understanding of Prx and the physiological role of StAhpC is discussed.

Fig. 1.

Reaction cycle of 2-Cys Prxs. The peroxidatic Cys of the Prx is depicted as a thiol (S_PH) or sulfenic acid (S_POH), or in a disulfide with the resolving Cys (S_RH). Colors distinguish the Cys residues from different subunits of the dimer in the typical 2-Cys Prxs, and the striped bar represents the intersubunit disulfide bond (intrasubunit in the case of the atypical 2-Cys Prxs). The disulfide reductase system that regenerates the active Prx varies with the organism and specific Prx but is Trx reductase and Trx in many eukaryotic systems and AhpF in the bacterial AhpC system. The pathway in the pink box represents regulation by oxidative inactivation (toward the right) and reactivation of hyperoxidized 2-Cys Prxs via sulfiredoxins (Srx) (toward the left).

Fig. 2.

Structures of the four hydroperoxide substrates for AhpC studied herein (Top and Middle) and an oxidized lipid substrate, a linoleic acid hydroperoxide (Bottom).

Fig. 3.

Differential activities of AhpC with two hydroperoxide substrates. Peroxidase activity was measured by mixing S128W NTD (prereduced by DTT) and AhpC (50–200 nM) in 50 mM potassium phosphate pH 7.0, 0.5 mM EDTA, and 100 mM ammonium sulfate with various concentrations of peroxide substrate in a stopped-flow spectrophotometer at 25°C. The reaction was followed by monitoring the decrease in fluorescence of S128W NTD (the modified NTD of AhpF, the direct electron donor to AhpC) with excitation at 280 nm and emission >320 nm. Fixed concentrations of S128W NTD used over a range of ethyl hydroperoxide (A) and t-butyl hydroperoxide (B) concentrations were 2.5 μM (■), 5 μM (○), 10 μM (▴), 15 μM (□), 20 μM (◆), and 30 μM (◇).

Fig. 4.

AhpC shows a susceptibility to inactivation by hydrogen peroxide and ethyl hydroperoxide, but not to t-butyl hydroperoxide and cumene hydroperoxide. NADPH oxidation was measured (by monitoring loss of 340-nm absorbance) at 25°C in the presence of 50 mM Hepes-NaOH, pH 7.0, with 1 mM EDTA and 0.1 M ammonium sulfate, and with 80 nM Trx reductase, 2.5 μM Trx, 6 μM AhpC and ethyl hydroperoxide (A) at 0 mM (●), 1 mM (■), 5 mM (□), 10 mM (▴), and 30 mM (◇). The same conditions were used to examine overoxidation with t-butyl hydroperoxide (B), except that concentrations of this substrate were at 0 mM (●), 2 mM (▾), 10 mM (▵), 20 mM (◆), and 60 mM (○). Results with hydrogen peroxide were very similar to those with ethyl hydroperoxide (A), whereas results with cumene hydroperoxide were quite similar to those with t-butyl hydroperoxide (B), except for issues with lower solubility and DMSO effects in the latter case.

Fig. 5.

HPLC profile of the separation of reduced and oxidized forms of StAhpC and E. coli Grx1. Reduced and oxidized AhpC and Grx1 (each at 50 μM) were allowed to equilibrate for 6 h at room temperature in 100 mM potassium phosphate, pH 7.0, with 1 mM EDTA. The protein mixtures were quenched with phosphoric acid and immediately separated by HPLC as described in Experimental Procedures. The red trace illustrates the mixture after oxidized Grx1 was equilibrated with reduced AhpC and is labeled to show where the redox forms of each of the proteins elute. The blue, dotted trace is displaced by 5 min and 0.05 absorbance units and shows the mixture after reduced Grx1 was equilibrated with oxidized AhpC.