The Use of Site-Directed Mutagenesis and the Ruthenium Modification Method for Study of Electron-Transfer Mechanisms in Metalloenzymes

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This project is in the Chemistry of Life Processes Initiative in the Office of Special Projects in Chemistry. It is jointly supported with the Biochemistry Program in the BBS Directorate. Advances in genetic engineering technology have provided the potential for designing novel proteins that offer convenient routes to the understanding of electron transfer mechanisms. By the use of site-directed mutagenesis this project will prepare optimized yeast iso-1 cytochrome c for the study of intramolecular electron-transfer mechanisms via the ruthenium modification method. The principal criterion for the selection of mutable sites is that the mutations be non-perturbative (that is each mutation should leave the system functionally identical with the wild-type system). The investigators have designed several mutants which will allow them to address the role of distance, medium, and driving force on long-range electron transfer. The distance study mutants cover a distance range of 12 to 18 Angstroms and are located on the protein's N-terminal alpha helix. The pathway mutants contain either aromatic or sulfur-containing residues in the electron-transfer pathways. In addition, studies on a double mutant will allow the possibility of through-bond electron transfer. Full driving force studies on ruthenated cytochromes at different electron-transfer distances will allow this project to separate electronic-coupling and nuclear-reorganization contributions to long-range electron-transfer rates.