Electron transfer reactions play key roles in a wide range of basic biological phenomena, including respiration, photosynthesis, nitrogen fixation and the biosynthesis of steroids and of DNA, as well as in more directly health related areas such as chemical carcinogenesis, drug detoxification, free radical damage and aging. This project has as its long-range goal the elucidation of the relationships between the molecular structures of the proteins involved in these processes and their biochemical and biophysical properties. The current proposal focusses on two aspects of this problem: i. electron transfer reactions occurring within transient protein-protein and protein-membrane complexes; ii. intramolecular electron transfer within multi-redox center proteins. In the first of these, the kinetics of electron transfer between two small redox proteins, ferredoxin and flavodoxin, and their physiological electron donors and acceptors, will be studied using laser flash photolysis methods. This technique allows direct measurements of the rates of the association step to form a productive electron transfer complex, and of the intracomplex electron transfer step, in structurally unmodified systems. As donors, the membrane-bound plant Photosystem I and the soluble human mitochondrial ferredoxin reductase will be utilized; acceptors include the soluble plant ferredoxin-NADP+ reductase and the membrane-associated mitochondrial cytochrome P450scc system. Site-specific mutagenesis of ferredoxins, flavodoxin and the reductases will be used to explore the roles of specific electrically charged and aromatic amino acid side chains in the processes of complex formation and electron transfer. In the area of multicenter redox proteins, attention will be focussed on the roles of charged and aromatic amino acid side chains, again using laser photolysis and site-specific mutagenesis techniques, in the intramolecular transfer of electrons between the flavin and heme cofactors of yeast flavocytochrome b2 (lactate dehydrogenase), and in the electron transfer between the b2 heme and the physiological acceptor cytochrome c. Additionally, studies of intramolecular electron transfer between the type I and type II,III copper centers of ascorbate oxidase and laccase will be carried out. It is anticipated that these studies will provide new insights into the roles of protein structure in controlling molecular recognition, specificity and rates in biological electron transfer processes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Biochemistry Study Section (BIO)
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University of Arizona
Schools of Arts and Sciences
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Feng, Changjian; Wilson, Heather L; Tollin, Gordon et al. (2005) The pathogenic human sulfite oxidase mutants G473D and A208D are defective in intramolecular electron transfer. Biochemistry 44:13734-43
Santagostini, Laura; Gullotti, Michele; Hazzard, James T et al. (2005) Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding. J Inorg Biochem 99:600-5
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Feng, Changjian; Wilson, Heather L; Hurley, John K et al. (2003) Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase. J Biol Chem 278:2913-20
Hurley, John K; Morales, Renaud; Martinez-Julvez, Marta et al. (2002) Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP(+) reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography. Biochim Biophys Acta 1554:5-21
Faro, Merche; Hurley, John K; Medina, Milagros et al. (2002) Flavin photochemistry in the analysis of electron transfer reactions: role of charged and hydrophobic residues at the carboxyl terminus of ferredoxin-NADP(+) reductase in the interaction with its substrates. Bioelectrochemistry 56:19-21

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