UC San Diego

Electron transfer is one of the most fundamental reactions observed in nature. Long-range electron transfer (ET) processes in biological systems often utilize aromatic amino acids as redox-active intermediates. Both tryptophan and tyrosine have been recognized as playing critical roles in mediating ET reactions that are essential to life, including photosynthesis and respiration. In contrast to the tyrosine radical, only a few studies have focused on the elusive tryptophan radical, which can exist in two distinct protonation states. One primary aim of this dissertation is the characterization of neutral tryptophan radicals within a protein scaffold to identify spectral markers of structure and local environment. Emphasis was placed on the use of resonance Raman spectroscopy, which has the capability to provide molecular-level detail but has been undervalued in this field. Azurin, a blue copper protein, is used as a model protein for the study of redox -active amino acids. Two neutral tryptophan radicals in different protein environments (ReAz108W* and Az48W*) were generated and characterized in detail via multiple spectroscopic techniques. The effects of hydrogen-bonding, environmental polarity, and geometry on absorption, EPR, and resonance Raman spectra of the radical were identified. Through this research, a spectroscopic library was established that correlates radical spectral parameters to local environment and structure. Rapid-flow mixing to generate the tryptophan cation radical enabled resonance Raman characterization of that species for the first time. Furthermore, a quantitative model for the photophysics of tryptophan radicals was developed. Study of the Az48W* radical revealed the ability of tryptophan to act as an excited-state reducing agent. Electron and proton transfer pathways within the protein were proposed and have begun to be probed using multiple techniques. Additionally, a related azurin variant was shown to demonstrate multistep ET between tryptophan, tyrosine, and the copper center on the sub-microsecond timescale. This system was investigated spectroscopically and biochemically to identify specific interactions that mediate these ET processes. It was found that the rapid proton-coupled electron transfer from tyrosine relies on intramolecular proton transfer to a nearby glutamate residue. These results have implications for understanding native systems and engineering multistep ET pathways within other proteins