Structure-Activity Studies of Surface Immobilized Cytochrome c Films on Indium Tin Oxide: Solution Adsorbed and Microcontact Printed Films

Abstract

The goal of this research project is to understand how different methods of protein immobilization affect the function and orientation of a redox active protein (cytochrome c) on an electrode surface. Cytochrome c (cyt c) is widely thought to adsorb onto negatively charged electrode surfaces with the heme pocket close to the electrode in a narrow orientation distribution that facilitates rapid electron transfer; however, a direct relationship between this orientation and the electron transfer properties has not been experimentally proven. Establishing structure-function relationships is crucial to the development of molecular devices based on immobilized proteins (e.g. biosensors). Differences in the structure and activity of two different types of protein films have been studied by comparing their surface coverage, orientation distributions and electrochemical behavior on the indium tin oxide (ITO) electrode surface. Films were formed by printing cyt c onto the ITO surface using microcontact printing (uCP) and by direct adsorption out of solution.A combination of surface-sensitive spectroscopic techniques was used to probe the prosthetic heme group of the protein, and by extension, the protein as a whole. Attenuated total reflectance (ATR) spectroscopy was used to measure the surface coverage and second order parameter of the cyt c films on glass and ITO. Differences in the surface coverage depended on the substrate and the method of deposition. Total internal reflectance fluorescence (TIRF) was used to measure the fourth order parameter of these films and the angle between the absorption and emission dipoles of the Zinc substituted heme. Using the second and fourth order parameters, orientation distributions were calculated using the maximum entropy method. The electrochemical behavior of these films was investigated using cyclic voltammetry. uCP films have a slightly slower rate of electron transfer and a lower electroactive surface coverage compared to films formed by solution adsorption. The percent of electroactive proteins in solution adsorbed films was about 50 %, while for uCP films it was only about 5 %.The ability to directly probe the total surface coverage and orientation distribution of molecules on the ITO electrode surface, as developed here, could be useful in many fields where optically transparent electrodes are in wide use, such as solar cells and organic light emitting diodes.