Engineering Pseudomonas aeruginosa Azurin for Energy and Electron Transfer

• Main Author: Tobin, Peter H
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01.01.2015
• Subjects: Biochemistry; Biophysics; Chemical engineering
• ISBN: 9781321946246; 1321946244

Electron transfer (EleT) and energy transfer (EngT) are common fundamental processes in life, and increasingly in materials engineering. Proteins involved in several life-critical processes including reaction centers in photosynthesis and photolyases in DNA repair have evolved protein matrixes with sophisticated temporal and spatial control of EleT and EngT. The ability to rationally design a protein matrix for EleT and/or EngT has not yet been fully realized, but would yield many benefits across bioenergetics, bioelectronics and biomedical engineering. Pseudomonas aeruginosa azurin has been an important model system for investigating fundamental EleT in proteins. Early pioneering studies used ruthenium photosensitizers to induce EleT in azurin and this experimental data continues to be used to develop theories for EleT mediated through a protein matrix. In this dissertation it is shown that putative EleT rates in the P. aeruginosa azurin model system, measured via photoinduced methods, can also be explained by an alternate EngT mechanism. Investigation of EngT in azurin, conducted in this study, isolates and resolves confounding phenomena—i.e., zinc contamination and excited state emission—that can lead to erroneous kinetic assignments. Extensive metal analysis, in addition to electrochemical and photochemical (photoinduced transfer) measurements suggests Zn-metallated azurin contamination can result in a biexponential reaction, which can be mistaken for EleT. Namely, upon photoinduction, the observed slow phase is exclusively the contribution from Zn-metallated azurin, not EleT; whereas, the fast phase is the result of EngT between the photosensitizer and the Cu-site, rather than simple excited state decay of the phototrigger. In order to circumvent the previously described problems with photoinduced measurements of EleT an orthogonal glassy carbon electrode based protein film voltammetry method was developed for measuring EleT rates in azurin. Finally, Computational Protein Design was utilized to modulate intramolecular EleT and EngT rates by engineering the residue composition in the core of azurin without perturbing the donor and acceptor sites.