Title

Protein redox reactions in thin films: Mono-oxygenase enzymes and photosynthetic reaction centers

Date of Completion

January 2003

Keywords

Chemistry, Analytical|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

This thesis addresses the development of novel stable layered thin films on electrodes containing mono-oxygenase enzymes and/or photosynthetic reaction center proteins for applications in sensors, enzymology, pharmacology and toxicology. Stable protein polyion, DNA and nanoparticle ultrathin films were developed using layer-by-layer self-assembly technique (chapters 2 and 3). Greatly enhanced electron transfer process, greater than 2 protein layers previously reported in our lab between Au electrodes and protein was obtained. ^ Electroactivity was extended in these films up to 7 layers in protein-polyion or DNA and up to 9 and 10 protein layers in protein nanoparticles (SiO 2, MnO2) films resulting in 7 to 17-fold increase in the amount of electroactive protein. Furthermore, Mb/DNA films (chapter 2) showed oxidation peaks after short incubations with styrene oxide that may be attributable to DNA damage. Results are relevant to the future design of enzyme-DNA films, which convert pollutants and drugs to toxic metabolites, followed by electrochemical detection of the resulting DNA damage. ^ Chapter 4 examines the applications of these protein polyion films to catalytic oxidation of pollutants, optimizing the peroxide mediated or electrochemical epoxidation of styrene using ultrathin films containing cyt P450cam and Mb. “Soft” ionic synthetic organic polymers like poly(styrene sulfonate), as opposed to SiO2 nanoparticles or DNA, support the best catalytic and electrochemical performance. Thin films (ca. 12–25 nm) gave the largest turnover rates for the catalytic epoxidation of styrene while thicker films were subject to reactant transport limitations. Classical bell-shaped activity-pH profiles and turnover rates similar to those in solution suggest that films grown layer-by-layer offer a new method to turnover rate studies of enzymes for organic oxidations. Major advantages include enhanced enzyme stability and the tiny amount of protein required. ^ Chapters 5, 6 and 7 explore thin film votammetry as a new tool in photosynthesis research. Chapter 5 explores electrochemical reactions of redox cofactors in purple bacteria Rhodobacter sphaeroides reaction center proteins in lipid films with peak assignment to the primary electron donor (P860) and quinone (QA) cofactors. Chapters 6 and 7 reports for the first time, direct, reversible electron transfer of cofactor redox sites in oxygenic photosystem I and II respectively in lipid films. These stable films may find applications in the construction of model biomedical devices, biosensors and bioreactors. Furthermore results are amenable to the future design of artificial photosynthesis. ^