Electrochemical, resonance Raman, and surface enhanced Raman spectroscopic study of biomolecules: cytochrome c and its mutants

Abstract

<p>In this work, conventional electrochemical methods were coupled with structure sensitive techniques, resonance Raman and surface-enhanced resonance Raman spectroscopies to study the electrochemical behavior, electron transfer mechanisms and configuration of the cytochrome c at modified metal electrode surfaces. Four studies were undertaken. The first one reevaluated two promoters which were studied by other research groups and a new electrode modification procedure was used. It was determined that both 2,2'-bipyridine and pyrazine act as promoters. The strengths of adsorption were found to be a very important factor which affects the promoter performance. A new idea about the structure requirement of the promoter was proposed. The second study found that thiophene, carbazole and pyridine whose molecules contain only one functional group, are all effective promoters which supported the previously proposed idea in the first study. The crucial role of these three promoters may be prevent denaturation and/or irreversible adsorption of cytochrome c or the deaminated and oligomeric components, which results in irreversible electrochemistry. In the third study, Quasi-reversible and direct electron transfer was observed between an iodide-modified gold electrode and cytochrome c. The results suggest that an electrostatic interaction between cytochrome c and the iodide-modified electrode surface plays an important role in the electrochemical response. Results obtained by surface enhanced resonance Raman scattering (SERRS) spectroscopy indicate that the heme group of the adsorbed cytochrome c is in the native low spin, six coordinate configuration at the iodide-modified Ag electrode, whereas at the bare Ag electrode a mixture of both low spin, six coordinate and high spin, five coordinate heme is present on the surface. In the forth study, several cytochrome c mutants were characterized by using cyclic voltammetry, resonance Raman and surface-enhanced resonance Raman spectroscopies to investigate how the mutations affect the redox potential, electron transfer kinetics and the stability of cytochrome. The results show that the mutations for the water mutants decrease the redox potentials which indicates that the mutation increases the stability of the protein. The RR results indicate that the heme environment is strongly perturbed by the mutation.</p>