Abstract
Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.
Highlights
Electron transfer involving various metabolites, external electron sources, and redox cofactors of proteins is a fundamental process in all domains of life on Earth
Molecular dynamics calculations provided a likely explanation for both the multiexponential behavior and for the distance dependence of the electron transfer rates. It appeared that the four-ring structure of the dye may occupy several positions close to the protein surface, stabilized by ionic interactions, and the electron may prefer a through space jump to or from the surface of the protein rather than the route following the covalent bonds connecting the dye to the labeled amino acid [18]
We studied the kinetics of electron transfer between the heme and the TUPSlabel, positioned at different sites on cytochrome c using the combined techniques of kinetic multichannel and single wavelength absorption spectroscopy
Summary
Electron transfer involving various metabolites, external electron sources, and redox cofactors of proteins is a fundamental process in all domains of life on Earth. High yield of the excited triplet state, and appropriate redox properties make the dye useful for initiation and analysis of electron transfer reactions in chemical and biological systems [7,8] The advantage of this system for electron transfer studies is its high efficiency; more than 20% of the protein molecules undergo intramolecular reduction in a single pulse. Molecular dynamics calculations provided a likely explanation for both the multiexponential behavior and for the distance dependence of the electron transfer rates It appeared that the four-ring structure of the dye may occupy several positions close to the protein surface, stabilized by ionic interactions, and the electron may prefer a through space jump to or from the surface of the protein rather than the route following the covalent bonds connecting the dye to the labeled amino acid [18]. Can bypass the covalent link, making TUPS a useful tool to study intra- and interprotein electron transfer processes
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