Electron Tunneling in Blue and Purple Copper Proteins

Citation

Hoke, Kevin Richard (2002) Electron Tunneling in Blue and Purple Copper Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/6WB9-2805. https://resolver.caltech.edu/CaltechTHESIS:01302012-102554225

Abstract

The CU_A domain is the initial point of entry for electrons into cytochrome c oxidase. In order to explain fast rates of intramolecular electron transfer (ET) over long distances, it has been suggested that the ET reorganization energy is less than 0.6 eV. This issue was investigated by attaching a ruthenium(II)-bisbipyridyl-imidazole complex to a single surface histidine of the Thermus thermophilus CU_A domain. Photoexcitation results in rapid ET between the ruthenium complex and the copper site. This was done over a range of driving forces by employing a series of substituted bipyridines. An analysis of the driving force dependence of the ET rate suggests that the total reorganization energy for this CU_A fragment is about 0.7 eV, almost as high as for azurin. The degree of solvent exposure of the active site may be the factor responsible for the higher reorganization energy.

In addition, the effect of distance on ET rate was investigated. Two variants of ruthenium-modified CU_A were examined, in which the labeled sites are two positions apart. The observed ET rates only differ by an order of magnitude, drastically less than what theory would predict. It appears that geometric factors other than length influence ET coupled through hydrogen bonds.

The role of hydrogen bonds in ET was studied in more detail with ruthenium-modified azurin. Changes in the ET rate were observed for different degrees of deuterium incorporation in the system. For wild-type azurin, the greatest effect (k_H/k_D ≈ 0.7) is seen when the protein is subjected to rigorous exchange under denaturing conditions, then returned to a normal buffer as a control for the solvent isotope effect on the reorganization energy. This is the expected outcome if deuteration of internal, difficult to exchange hydrogen bonds results in improved coupling along the path.

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