Intramolecular Electron Transfer and Conformational Changes in Cytochrome c Oxidase

Abstract

The photolysis intermediates of partially and fully reduced CO-bound cytochrome oxidase derivatives were investigated. A gated optical spectrometric multichannel analyzer was used to collect visible and near-infrared transient difference spectra on time scales from nanoseconds to milliseconds. The spectra were analyzed by a singular value decomposition method combined with a global exponential fitting procedure. Global analysis of the mixed-valence CO complex transient difference spectra shows that five intermediates are present with apparent lifetimes of 1.4 microseconds, 4.8 microseconds, 76.7 microseconds, 10.6 ms, and 21.6 ms. The data were fitted to a kinetic model involving a sequential pathway with accompanying equilibria. On the basis of this mechanism, the absorption spectra of the intermediates were determined. The first step, also present in the fully reduced enzyme, is attributed to a conformational change at cytochrome a3. The spectral changes associated with the second step are similar to those expected for 1:1 electron transfer from cytochrome a3 to cytochrome a, except for a higher absorbance between 480 and 550 nm. A comparison of the experimental spectral change associated with this step, (a2+ minus a3+) minus (a3(2+) minus a3(3+), and the calculated spectral change, (a2+ CuA+ minus a3+ CuA2+) minus a3(2+) CuB+ minus a3(3+) CuB2+), allowed extraction of the absorbance spectrum of CuA2+ in the 480-550 nm region. The spectral change associated with the third step is consistent with the oxidation of cytochrome a. A decrease in the 830 nm band on the same time scale indicates that the electron acceptor is CuA. The data also suggest that the redox state of CuB significantly affects the absorption spectrum of oxidized cytochrome a3 in the visible region. The two processes on a millisecond time scale are attributed to CO recombination and intramolecular electron transfer.

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain

“…As shown in Fig. 1A we observed the characteristic derivative-shaped spectrum reflecting the photo-dissociation of CO and the resulting band shift of the Soret band of the reduced heme a 3 from 430 nm to 445 nm (31,32). We note however that this difference spectrum was slightly red-shifted (by c.a.…”

The Responses of Cytochrome Redox State and Energy Metabolism to Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation Tolerance

“…The asterisk indicates spectral features characteristic of a rigid-solid-state-like spectrum. to O 2 and proton pumping (Einarsdottir et al, 1995;Wittung and Malmströ m, 1996). When the extent of COX reduction in cowpea cotyledons and T. latifolia pollen were plotted against viscosity, a similar relation appeared for both materials despite their differences in cellular composition and structure (Fig.…”

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

“…However, it is a non-trivial task to study the forward electron transfer reactions because they are tightly coupled with the oxygen reduction chemistry. On the other hand, flash photolysis of the so-called mixed valence CO-bound derivative (MVCO) has been extensively used to study the reverse electron transfer reactions in CcO (5)(6)(7)(8).…”

“…It is followed by an electron transfer reaction from heme a 3 to heme a that leads to species c. The subsequent electron transfer from heme a to Cu A produces species d. Each step in the electron transfer chain described in Scheme 1 is reversible. In addition, it is believed that one electron always resides on Cu B because it is proposed to have a midpoint potential that is much higher than those of the two hemes (6,8).…”

“…In addition to the fast phases, millisecond time scale changes were also observed that were ascribed to the structural rearrangement associated with the electron redistribution and/or CO recombination (6,7). Based on optical absorption spectroscopy, Einarsdottir and co-workers (5, 6) pointed out that in addition to the four species included in Scheme 1, other intermediates have to be considered to account for the changes observed in the optical spectra.…”