A great many biological energy transduction pathways depend upon the rapid movement of electrons or holes over long distances (>30 ?) through proteins. Many redox enzymes, particularly those involved in oxygen activation and production, require the transfer of holes at high potentials where the sidechains of redox-active amino acids can become involved. Stringent design requirements must be met to transport charges rapidly and efficiently along specific pathways and prevent the off- path diffusion of redox equivalents and the disruption of energy flow. In this research program, the molecular parameters that control multistep electron tunneling through tryptophan (Aim 1) and tyrosine (Aim 2) radicals will be examined. Several azurin mutants in which tryptophan residues lie between the Cu active site and a surface-attached photosensitizer will be prepared. Kinetics measurements will be used to elucidate the roles of redox-site separation distance, driving force, and tryptophan environment on the dynamics of long-range charge transport. Tyrosine is postulated to mediate long-range hole transport in several redox enzymes. Azurin mutants in which tyrosine residues are placed to serve as mediators of long-range electron transfer will be prepared and studied. Mutant proteins in which proton accepting residues are within hydrogen-bonding distance of the intermediate tyrosine residue also will be prepared. These proximal proton acceptors are expected to facilitate long-range hopping via tyrosine radicals. Amino-acid sidechain radicals often are found in enzymes involved in oxygen activation and formation chemistry. Important members of this class of enzymes are the heme-thiolate mono- oxygenases such as cytochrome P450 and nitric oxide synthases (NOS). These enzymes utilize molecular oxygen to effect a wide range of hydroxylation reactions involving organic substrates. Despite extensive investigations in many laboratories, questions remain regarding the nature of the active hydroxylating reagent in these enzymes: ferryl species (i.e., Fe-oxo Compounds I and II) are often invoked. Protein labeling and laser flash-quench photochemistry will be used to develop new insights into the reactive intermediates in heme-thiolate mono-oxygenases. Rather than treating the enzymes with reactive oxygen compounds, photochemically generated one-electron, outer-sphere oxidants covalently bound to the protein periphery will be used to remove electrons from the resting enzyme and produce ferryl compounds. Efforts will focus on oxidative generation of Compounds I and II in cytochromes P450 (Aim 3) and a bacterial enzyme that exhibits NOS-like reactivity (Aim 4).

Public Health Relevance

Reduction and oxidation (redox) reactions are vital transformations in a wide array of metabolic processes. Countless diseases are associated with failures or disruptions of redox pathways. Elucidation of the fundamental chemical factors that control biological redox processes will lead to deeper understanding of disease mechanisms and guide the development of new therapies.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Sechi, Salvatore
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California Institute of Technology
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United States
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Saouma, Caroline T; Pinney, Margaux M; Mayer, James M (2014) Electron transfer and proton-coupled electron transfer reactivity and self-exchange of synthetic [2Fe-2S] complexes: models for Rieske and mitoNEET clusters. Inorg Chem 53:3153-61
Gray, Harry B; Warren, Jeffery J; Winkler, Jay R et al. (2014) A Euclidean perspective on the unfolding of azurin: chain motion. J Biol Inorg Chem 19:555-63
Winkler, Jay R; Gray, Harry B (2014) Electron flow through metalloproteins. Chem Rev 114:3369-80
Winkler, Jay R; Gray, Harry B (2014) Long-range electron tunneling. J Am Chem Soc 136:2930-9
Pospíšil, Petr; Luxem, Katja E; Ener, Maraia et al. (2014) Fluorescence quenching of (dimethylamino)naphthalene dyes Badan and Prodan by tryptophan in cytochromes P450 and micelles. J Phys Chem B 118:10085-91
Cable, Morgan L; Kirby, James P; Gray, Harry B et al. (2013) Enhancement of anion binding in lanthanide optical sensors. Acc Chem Res 46:2576-84
Yamada, Seiji; Bouley Ford, Nicole D; Keller, Gretchen E et al. (2013) Snapshots of a protein folding intermediate. Proc Natl Acad Sci U S A 110:1606-10
Bouley Ford, Nicole D; Shin, Dong-Woo; Gray, Harry B et al. (2013) Intrachain contact dynamics in unfolded cytochrome cb562. J Phys Chem B 117:13206-11
Warren, Jeffrey J; Gray, Harry B; Winkler, Jay R et al. (2013) A Euclidean Perspective on the Unfolding of Azurin: Spatial Correlations. Mol Phys 111:922-929
Warren, Jeffrey J; Winkler, Jay R; Gray, Harry B (2013) Hopping Maps for Photosynthetic Reaction Centers(). Coord Chem Rev 257:165-170

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