Modulation of the Electron Redistribution in Mixed Valence Cytochrome c Oxidase by Protein Conformational Changes

Abstract

The redistribution of two electrons in the four redox centers of cytochrome c oxidase following photodissociation of CO from the CO-bound mixed valence species has been examined by resonance Raman spectroscopy. To account for both the kinetic data, obtained from 5 s to 2 ms, and the equilibrium results, a model is proposed in which the electron redistribution is modulated by a protein conformation transition from a nascent P 1 state to a relaxed P 2 state in a time window longer than 2 ms. In this model, all six possible two-electron reduced species are considered. The high population of species with a one-electron reduced binuclear center, in which the spectrum of heme a 3 is perturbed by the redox state of Cu B , accounts for the significant residuals in the fitting of the kinetic data with four standard spectra derived from redox species with either zero or two electrons in the binuclear center. Under equilibrium conditions, the conformational change to the P 2 state destabilizes the redox states with only one electron in the binuclear center with respect to those with either zero or two electrons. As a result, the redox equilibrium is perturbed, and the electrons are redistributed. A simulation based on the new kinetics scheme, in which the electron redistribution is modulated by the protein conformation, gives reasonable agreement with both the equilibrium and the kinetic data, demonstrating the validity of this model.

Electron transfer pathways in cytochrome c oxidase

“…In the heme a 3 -Cu B site case there is a larger degree of delocalization, as it had been observed by the spin density plots, due to the close proximity of the redox centers. In agreement with our findings the perturbed heme a 3 spectra observed by Ji et al [60] could be explained by the delocalization of this single electron between the heme a 3 and Cu B centers. This one electron reduced state, termed the E state, has been reported to have an electron distribution among the redox centers [63–65].…”

“…Concerning the heme a 3 , three situations have been explored: low spin (equivalent to the heme a ) and high spin, both coupled and uncoupled to the copper Cu B atom. According to experimental evidence [59, 60] the iron atom should exhibit a high spin state but our calculations show that the lowest energy state to be the low spin. However, care must be taken as the theory level seems to play an important role in accessing the correct spin state.…”

Reaction Mechanism of Cytochrome c Oxidase

“…By analogy to the CO-binding, the complete reduction of the O 2 -reduction site is very likely to be prerequisite for O 2 binding. The infrared spectrum and X-ray structure of CO at Cu B indicate a fairly weak (essentially side-on) binding without any significant interaction with Fe a 3 . , However, the small but significant spectral changes in the 1 μs phase described above indicate that some significant interactions exist between Cu B and Fe a 3 , even when both metals are in the unliganded reduced state. A significant enhancement of electron transfer to Fe a 3 by anaerobic reduction of Cu B was revealed by stopped-flow freeze-quench EPR spectroscopic analyses The influence of the oxidation state of Cu B to the oxidized heme a 3 absorption spectrum has been confirmed by an extensive resonance Raman analyses The CO-stretch band of CO bound to Cu B shifts from 2061 to 2040 cm –1 upon oxidation of Fe a 3 …”

Structural characterization of the PCO/O2 compound of cytochrome c oxidase

“…The Raman measurements were performed with previously described instrumentation [13]. The excitation source was a He-Cd laser with an output of 441.6 nm.…”