Radical formation and electron transfer in biological molecules

Abstract

Multi-step electron transfer is increasingly recognized as a means for moving charge in biological systems over long distances rapidly. Many postulated multi-step mechanisms rely on the formation of organic radicals (amino-acid radicals, nucleobase radicals) as intermediate electron or ?hole? carriers. In this thesis, the multi-step mechanism for electron transfer in both proteins and DNA is investigated. These two systems form a natural complement; the role of electron transfer in DNA with regard to lesion repair is still unknown, as are the electron transfer events in the proteins that mediate the repair. Rhenium-labeled mutant Pseudomonas aeruginosa azurins serve as model systems for this phenomenon in proteins. The photo-active rhenium label in these azurins can be oxidized by a flash/quench reaction to provide a potent oxidant capable of generating a variety of radicals in the protein matrix. Three mutants (with one tryptophan residue each) were constructed to investigate the effect of tryptophan radicals on charge transfer in proteins. The properties of tryptophan radicals in three protein environments have been investigated; including a kinetically stable tryptophan radical that persists for more than 5 hrs at room temperature. The variation in these radicals plays a significant role in their effect on the oxidation of the remote copper center in azurin. The stable radical greatly reduces the rate of electron transfer from copper relative to the rhenium-labeled wild-type analogs, while another radical plays no role in copper oxidation. In order to examine multi-step electron transfer in DNA, a series of photo-active ruthenium and rhenium-thymine complexes were constructed. By attaching the metal complexes at the sugar of the deoxyribonucleic acid, they were incorporated into DNA strands by solid-state synthetic techniques. Two different ruthenium-labeled DNA strands were produced in this way; one with a single guanine base and one with two side by side guanine bases. The strand containing a guanine-guanine sequence showed formation of a guanine radical by EPR under flash/quench conditions, while the strand containing a single guanine remained EPR silent. These strands represent an excellent template to examine a system which may or may not exhibit multi-step charge transfer.