DIRECT ELECTROCHEMICAL ANALYSIS OF THE REDOX ACTIVITY OF TRYPTOPHAN AND TYROSINE IN MODIFIED AZURINS: THE IMPACT OF THE PROTEIN ENVIRONMENT

Abstract

Proton-coupled electron transfer (PCET) is a biological process essential to life. It is imperative for respiration in animals as well as photosynthesis in plants. Long-range PCET is often facilitated by redox-active amino acids, such as tryptophan and tyrosine. While there are several examples in the literature for the involvement of these redox-active residues in PCET linked to biological catalysis, there has been a challenge in direct electrochemical efforts to resolve how the local protein environment controls PCET directionality. This thesis describes a protocol from which to directly test the reduction potentials of tyrosine and tryptophan radicals in a customizable protein environment. The model protein used for this study was azurin, a natural cupredoxin that natively contains two tyrosines and only one tryptophan, with the former mutated to phenylalanine to provide direct electrochemical detection of a single redox-active amino acid species. The reduction potentials of azurin with either tryptophan or tyrosine redox centers were monitored using the electrochemical technique square-wave voltammetry. Using this technique along with strategic protein engineering, it was found that the more solvent-exposed or polar mutants had a higher redox potential than those that were more solvent-excluded. These trends have biological importance as the difference in the reduction potentials between redox-active amino acid pairs is expected to control the thermodynamic driving force for PCET. This thesis details the impact of altering the surrounding protein environment, i.e. electrostatics, on the redox activity of tryptophan and tyrosine.