Mechanistic studies of alkyl hydroperoxide reductase

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The bacterial alkyl hydroperoxide reductase system serves to protect against the toxic and mutagenic effects of oxidative stress. AhpC, the cysteine-based peroxidase component, is a member of the ubiquitous "peroxiredoxin" (Prx) family and reduces H2O2 and organic hydroperoxides through transient generation of a cysteine sulfenic acid on the enzyme and subsequent intersubunit disulfide bond formation. AhpF, the flavin-containing reductase component, is present in most, but not all, bacteria and efficiently transfers electrons from NADH (or NADPH) to AhpC. Mammalian Prxs have been implicated in such diverse processes as cellular proliferation and differentiation, immune responses and cell signaling. While most AhpC/Prx homologues are highly expressed and play an important role in oxidative defense, only the AhpC from Helicobacter pylori (the causative agent of gastric ulcers linked to stomach cancer) is known to be absolutely required for viability of that organism. The first specific aim of the proposal focuses on (1) the conformational states, oligomerization and membrane association thought to change during turnover of Salmonella typhimurium AhpC and mammalian Prx II in the presence of peroxides, and (2) the participation of a putative general base catalyst (Arg119) in peroxide reduction by AhpC. The second specific aim explores the mechanism of electron transfers to and from the N-terminal disulfide center of S. typhimurium AhpF. This center (Cys129- Cys132) is part of a distinct redox domain in AhpF known from our studies to mediate electron transfer from redox centers (FAD and Cys345-Cys348) in the C-terminal portion of the protein to AhpC. Our recent crystallographic analyses of AhpF have demonstrated a unique architecture for the N-terminal domain (NTD) and a poorly- characterized homologue, protein disulfide oxidoreductase (PDO), from a thermophile; both NTD and PDO are composed of two intimately-associated thioredoxin-like folds with a putative active site glutamate from the first half acting as a general acid-base catalyst for chemistry at the Cys-X-X-Cys motif of the second half of the domain. Our crystallographic analyses of AhpF also strongly support the involvement of large domain movements in the catalytic cycle of AhpF. Crystallographic and fluorescence approaches will be used in the third specific aim to define the nature of AhpF-AhpC interactions as well as inter- domain interactions within AhpF during intrasubunit electron transfer. Understanding of catalysis by bacterial AhpF and both bacterial and mammalian AhpC homologues will contribute to our knowledge of oxidative stress defense mechanisms and redox-regulated cell signaling in both pathogens and mammalian hosts. Therapeutic intervention in preventing oxidative damage involved in human degenerative diseases, cancer and aging as well as in combating pathogenic defense systems requires a complete molecular and biological understanding of the alkyl hydroperoxide reductase enzymes from both bacterial and human sources.