UC San Diego

Amino acid radicals are important for electron transfer reactions in biological systems. Fundamental processes such as cellular respiration, photosynthesis and magnetoreception of migratory species rely on amino acid radicals to facilitate long distance electron transfer. The tryptophan residue in modified Zn-azurin protein is known to form a stable neutral radical through a photoinduced proton coupled electron transfer reaction in the presence of an exogeneous electron acceptor. A goal of this dissertation is to elucidate the photophysical mechanisms that produces the long-lived neutral radical in Zn-azurin using electronic spectroscopy and to determine the effects of deuteration to the Raman vibrational spectrum and the photoinduced proton coupled electron transfer reaction. The results of a spectroscopic study of the photoinduced proton coupled electron transfer reaction that generates a stable tryptophan neutral radical in Zn-azurin are presented. The quantum yields for electron transfer, tryptophan neutral radical formation, fluorescence, phosphorescence, and the efficiency of proton transfer are reported for Zn-azurin in the presence of the electron acceptors cobalt(III)pentaammine chloride and Cu(II)-azurin. The electron transfer and tryptophan neutral radical quantum yields increased with increasing concentrations of electron acceptor, however, the phosphorescence quantum yield decreased and the fluorescence quantum yield was unaffected. These results indicate that the triplet state is the parent state of electron transfer. A Stern-Volmer analysis of phosphorescence quenching revealed that cobalt(III)pentaammine chloride (KSV = 230,000 M-1) is a more effective quencher than Cu(II)-azurin (KSV = 88,000 M-1). The KSV for Cu(II)-azurin is consistent with reported values for dynamic quenching of tryptophan in azurin, however the KSV for cobalt(III)pentaammine chloride is larger than the reported values for dynamic quenching with smaller charged molecules. Samples containing both electron acceptors indicate that cobalt(III)pentaammine chloride is the preferred electron acceptor. More efficient and preferential quenching with cobalt(III)pentaammine chloride suggests that the quenching mechanism could be static in nature. The calculated and experimental resonance Raman spectra of tryptophan and perdeuterated tryptophan, in solution, in azurin, and as the photogenerated neutral radical in azurin are described. The vibrational modes of the deuterated isotopologue are generally lower in frequency compared to protiated tryptophan and the resonance Raman intensities vary; these differences likely reflect changes in the normal mode compositions of the isotopologue. Despite these changes, several of the perdeuterated tryptophan vibrational modes retain their ability to report on environmental factors such as hydrogen bonding and conformation of the indole ring. Resonance Raman spectra of the perdeuterated tryptophan neutral radical in azurin had lower frequencies and different intensities from the protiated isotopologue. Furthermore, the quantum yield of the perdeuterated neutral radical was lower, possibly due to changes in vibrational modes that are coupled to the electron transfer. The data and analyses presented here deepen our understanding of the mechanisms and properties of the tryptophan residue in azurin in the context of electron transfer. The results highlight the wealth of information that can be obtained with spectroscopic tools combined with an isotopologue. These findings inspire new experiments to further enhance our knowledge of tryptophan radicals in long-range electron transfer.