A Kinetic Model of Proton Transport in a Multi-Redox Centre Protein: Cytochrome c Oxidase

Abstract

We use chemical reaction kinetics to explore the stepwise electron and proton transfer reactions of cytochrome c oxidase (CcO) from R. sphaeroides. Proton transport coupled to electron transport (ET) is investigated in terms of a sequence of protonation-dependent second-order redox reactions. Thereby, we assume fixed rather than shifting dissociation constants of the redox sites. Proton transport can thus be simulated particularly when separate proton uptake and release sites are assumed rather than the same proton pump site for every ET step. In order to test these assumptions, we make use of a model system introduced earlier, which allows us to study direct ET of redox enzymes by electrochemistry. A four-electron transfer model of CcO had been developed before, according to which electrons are transferred from the electrode to Cu A . Thereafter, electrons are transferred along the sequence heme a, heme a 3 and Cu B . In the present investigation, we consider protonation equilibria of the oxidised and reduced species for each of the four centres. Moreover, we add oxygen/H 2 O as the terminal (fifth) redox couple including protonation of reduced oxygen to water. Finally we arrive at a kinetic model comprising five protonation-dependent redox couples. The results from the simulations are compared with experimental data obtained in the absence and presence of oxygen. As a result, we can show that proton transport can be modelled in terms of protonation-dependent redox kinetics.

Time-Resolved Surface-Enhanced IR-Absorption Spectroscopy of Direct Electron Transfer to Cytochrome c Oxidase from R. sphaeroides

“…In our model system developed previously (19,21), we employ chemical reaction kinetics to describe the sequential ET between redox centers, indicated by black arrows, which can each be in the reduced and oxidized state, denoted by r and o, respectively ( Fig. 1) Proton uptake and release ( Fig.…”

“…Absorbances plotted as a function of time will then be fitted to the extended model mentioned above (21). Fit parameters will be compared with those obtained in modeling studies of electrochemical data (19,21). Our results suggest that conformational changes coupled to redox transitions occur on the millisecond timescale dominated by the rate constant of electrochemical excitation.…”

“…Two-dimensional-correlation maps of these SEIRA spectra showed substantial conformational changes of the CcO as it is converted from the nonactivated to the activated state (20). The sequential four-ET model was later expanded to describe proton transport coupled to ET, taking into consideration acid dissociation constants of the oxidized and reduced forms (pKo and pKr, respectively) of all four redox centers (21).…”

“…Based on comparative IR and UV/VIS data found in the literature (23), the deconvoluted components will be attributed to changes in specific redox centers. Absorbances plotted as a function of time will then be fitted to the extended model mentioned above (21). Fit parameters will be compared with those obtained in modeling studies of electrochemical data (19,21).…”

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS

“…Protons arriving within the small submembrane space (~0.2 nL cm -2 ) as a result of enzyme activity can be electrochemically reduced to hydrogen [7,12]. The main advantage, however, lies in the fact that stepwise ET can be investigated electrochemically in the presence and absence of oxygen [13,14]. In order to analyze the results of fast-scan voltammetry of the CcO, we made use of an electrochemical four electron transfer model of CcO displayed in Schematic 2.…”