Research Interests

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.

More on Crystallographic Studies with Drs. Zac Wood and Andy Karplus

Recent Selected Publications

4/25/2003 Science Article Press Releases:

WFU PRESS RELEASE, Science article, : Study of Bacterial Enzyme Reveals One Key to Cancer Cell Survival

BBC News Online

Gazette Times (Corvallis, OR)

Chemical & Engineering News

Faculty of 1000 "Must Read" summary

Other Publications

Poole, L.B., and Ellis, H.R.  (1996) Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 1. Purification and enzymatic activities of overexpressed AhpF and AhpC proteins. Biochemistry 35, 56-64. PDF of article

Poole, L.B.  (1996) Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction. Biochemistry 35, 65-75. PDF of article

Poole, L.B., Chae, H.Z., Flores, B.M., Reed, S.L., Rhee, S.G., and Torian, B.E.  (1997) Peroxidase activity of a TSA-like antioxidant protein from a pathogenic amoeba. Free Radical Biol. Med. 23, 955-959. PDF of article

Li Calzi, M., and Poole, L.B. (1997) Requirement for the two AhpF cystine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase. Biochemistry 36, 13357-13364. PDF of article

Ellis, H. R., and Poole, L.B. (1997) Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium.Biochemistry 36, 13349-13356. PDF of article

Ellis, H. R., and Poole, L.B. (1997) Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36, 15013-15018. PDF of article

Poole, L.B. (1997) The Salmonella typhimurium alkyl hydroperoxidereductase enzyme system, in Flavins and Flavoproteins 1996 (K.J. Stevenson,V. Massey and C.H. Williams, Jr., eds) University of Calgary Press, Calgary,Alberta, Canada, 751-760.

Higuchi, M., Yamamoto, Y., Poole, L.B., Shimada, M., Sato, Y., Takahashi,N., and Kamio, Y. (1999) Functionsof two types of NADH oxidases in energy metabolism and oxidative stressof Streptococcus mutans. J. Bacteriol. 181, 5940-5947. PDFof article

Poole, L.B.  (1999)  Flavin-linked redox components requiredfor AhpC reduction in alkyl hydroperoxide reductase systems.  In Flavinsand Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux and H. Sund,eds.) Agency for Scientific Publications, Berlin, Germany, 195-202.

Reynolds, C.M., and Poole, L.B.  (1999)  Functional characterizationof the N-terminus of AhpF by chimeric construction with TrR.  In Flavinsand Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux and H. Sund,eds.) Agency for Scientific Publications, Berlin, Germany, 681-684.

Higuchi, M., Yamamoto, Y., Poole, L.B., Shimada, M., Sato, Y., Takahashi,N., and Kamio, Y.  (1999)  Functional and regulatory studiesof two distinct NADH oxidases from Streptococcus mutans.  InFlavins and Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux andH. Sund, eds.) Agency for Scientific Publications, Berlin, Germany, 691-694.

Poole, L.B., Higuchi, M., Shimada, M., Li Calzi, M., and Kamio, Y. (2000) Streptococcus mutans H₂O₂-forming NADH oxidase is an alkyl hydroperoxide reductase protein.  Free Radical Biol. Med. 28, 108-120. PDF of article

Poole, L.B., Godzik, A., Nayeem, A. and Schmitt, J.S.  (2000) AhpF can be dissected into two functional units: tandem repeats of twothioredoxin-like folds in the N-terminus mediate electron transfer fromthe thioredoxin reductase-like C-terminus to AhpC. Biochemistry 39,6602-6615. PDFof article, PDFof supporting info

Reynolds, C.M., and Poole, L.B. (2000) Attachmentof the N-terminal domain of Salmonella typhimurium AhpF to Escherichiacoli thioredoxin reductase confers AhpC reductase activity but doesnot affect thioredoxin reductase activity. Biochemistry 39, 8859-8869.PDFof article, PDFof supporting info

Yamamoto, Y., Higuchi, M., Poole, L.B., and Kamio, Y.  (2000) Identificationof a new gene responsible for the oxygen tolerance in aerobic life of Streptococcusmutans.  Biosci. Biotechnol. Biochem. 64, 1106-1109.

Yamamoto, Y., Higuchi, M., Poole, L.B., and Kamio, Y.  (2000) Role of the dpr product in oxygen tolerance in Streptococcus mutans.  J. Bacteriol. 182, 3740-3747. PDF of article

Poole, L.B., Reynolds, C.M., Wood, Z.A., Karplus, P.A., Ellis, H.R. and Li Calzi, M. (2000) AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase. Eur. J. Biochem. 267, 6126-6133. PDF of article

Baker, L.M.S., Raudonikiene, A., Hoffman, P.S., and Poole, L.B. (2001) An essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization.  J. Bacteriol. 183, 1961-1973. PDF of article

Reynolds, C.M., and Poole, L.B. (2001) Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains.  Biochemistry 40, 3912-3919.  PDF of article

Wood, Z.A., Poole, L.B., and Karplus, P.A. (2001) Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis.  Biochemistry 40, 3900-3911. PDF of article

Reynolds, C.M., Meyer, J., and Poole, L.B. (2002) An NADH-dependent bacterial thioredoxin reductase-like protein, in conjunction with a glutaredoxin homologue, form a unique peroxiredoxin (AhpC) reducing system in Clostridium pasteurianum. Biochemistry 41, 1990-2001. PDF of article, PDF of supporting material

Conway, M.E., Yennawar, N., Wallin, R., Poole, L.B., Hutson, S.M. (2002) Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase. Biochemistry 41, 9070-9078. PDF of article.

Yamamoto, Y., Poole, L.B., Hantgan, R.R., and Kamio, Y. (2002) An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro. J. Bacteriol. 184, 2931-2939. PDF of article.

Conway, M.E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S.M. (2003) Human mitochondrial branched chain aminotransferase: Structural basis for substrate specificity and role of redox active cysteines. Biochim. Biophys. Acta 1647: 61-65. PDF of article.

Schröder, E., Jönsson, T., and Poole, L. (2003) Hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of pH. Anal. Biochem. 313: 176-178. PDF of article.

Biographical Information for Dr. Poole

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