Organisms living in aerobic environments require mechanisms which prevent or limit damage to cellular components by reactive oxygen species; these species arise from the incomplete reduction of oxygen during respiration or from exposure to external agents such as light, radiation, redox-cycling drugs or stimulated host phagocytes. A variety of enzymatic and nonenzymatic systems have evolved within living organisms to counter such damage. In bacteria such as Salmonella typhimurium and Escherichia coli, expression of a set of antioxidant enzymes is controlled in a coordinate fashion by an oxidation- reduction-sensitive regulatory protein, OxyR. Using a high-expression OxyR mutant of S. typhimurium, a novel enzymatic activity responsible for the NAD(P)H-linked reduction of toxic organic hydroperoxides was discovered. This alkyl hydroperoxide reductase (AhpR), which is the focus of studies in my laboratory, was subsequently shown to be separable into two protein components, designated AhpF and AhpC. AhpF is an FAD-containing protein related to another well- characterized flavoprotein, thioredoxin reductase, and catalyzes the transfer of electrons from NAD(P)H to AhpC. The smaller AhpC protein is without a chromaphoric cofactor and serves directly as the peroxide-reducing component (homologues of AhpC are widespread in biological systems and have been designated "peroxiredoxins"). Our studies of the catalytic mechanism of AhpR indicate that both component proteins operate through cycling of protein- derived cystine disulfides between oxidized (disulfide) and reduced (dithiol) states. Anaerobic titrations of each protein with reductants have confirmed the presence and essentiality of three redox centers in AhpF (one FAD and two disulfide centers) and one redox-active disulfide center per monomer in AhpC (Poole, 1996; Poole, Godzik, et al, 2000). We have also shown that an unusual oxidized cysteine derivative, cysteine-sulfenic acid (Cys-SOH), is generated transiently through direct oxidation of Cys46, one of the two cysteine residues of AhpC, by the hydroperoxide substrate (Ellis & Poole, 1997a & b). Related oxidized cysteine derivatives have been identified in the two other known heme- and metal-independent peroxide reductases (NADH peroxidase and glutathione peroxidases) and may play a role in signal transduction by the OxyR protein itself.
Studies of the structural and chemical bases for the enzymatic functions of AhpF and AhpC involve a wide range of biochemical techniques. Site-directed and PCR-based mutagenesis experiments have been designed to address specific structure-function questions. Characterization of native and mutant proteins involve enzymological techniques, such as spectral titrations and rapid reaction kinetic measurements, protein chemistry methodology to define cysteine content and redox status, and structural work, which includes protein crystallization and analytical ultracentrifugation.