Redox Kinetics Of Cytochrome C Oxidase By Electrochemically-induced Time-resolved Surface-enhanced Infrared Absorption Spectroscopy (tr-Seiras)

Abstract

For use with surface-enhanced infrared absorption spectroscopy, SEIRAS, we designed a two-layer gold surface for use with electrochemistry which consists of a conducting underlayer onto which Au nanoparticles (AuNPs) are grown by self-catalyzed electroless deposition. This enabled us to apply time-resolved (tr)-SEIRAS to cytochrome c oxidase (CcO) from R. sphaeroides immobilized in a strictly oriented fashion on the two-layer gold surface. The enzyme was excited by direct electron transfer (electronic wiring) to the CuA redox center. Electrons then travelled through the rest of the electron transfer chain to heme a, heme a3 and CuB without using any mediators. Kinetic constants were obtained by applying periodic potential pulses and recording spectral changes as a function of time. Excitation frequencies were varied in a wide range between 0.7 Hz and 2 kHz. Time resolved spectra were analyzed by phase-sensitive detection. A wide range of kinetic constants was obtained thereby separating these parameters from the contribution due to charging currents.Tr-SEIRA spectra of the cytochrome c oxidase at different excitation frequencies10 Hz 250 HzCh. Nowak, Ch. Luening, D. Schach, D. Baurecht, W. Knoll, R. L. C. Naumann, electron transfer kinetics of cytochrome c in the sub-ms time regime using time-resolved SEIRAS, JPC(C) under review.