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 ( St AhpC), 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 St AhpC ( k cat / K m ) are >10 7 M −1 ·s −1 for inorganic and primary hydroperoxide substrates and ≈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 St AhpC 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 St AhpC is discussed.