Conformational changes, internal electron transfer, and CO rebinding processes in cytochrome c oxidase from Rhodobacter sphaeroides reduced to different degrees were investigated. The reactions were followed using a gated optical spectrometric multichannel analyzer. Light-induced difference spectra, recorded in the 350-700 nm region over the 100 ns to 1 s time interval, were analyzed by singular value decomposition and global exponential fitting. The photolyzed fully reduced enzyme showed two relaxations, ∼1 and 190 μs, prior to the 20 ms CO rebinding process. Intramolecular electron transfer was monitored following photolysis of the mixed-valence CO-bound enzyme. The analysis revealed 1.1 μs, 2.4 μs, 31 μs, 68 ms, and 240 ms apparent lifetimes, the first three of which are attributed to electron transfer from heme a3 to heme a with contribution from a relaxation process at the heme a3 site. Spectral changes associated with the microsecond processes are consistent with 75% electron transfer from heme a3 to heme a. A comparison of the experimental spectra and model difference spectra for the intramolecular electron transfer indicated ∼3 nm blue shift in the absolute spectra of both the oxidized heme a3 and reduced heme a generated in the process. The 68 and 240 ms lifetimes are due to CO recombination to heme a3 and are attributed to the presence of two conformers, the slower rate corresponding to the conformer in higher abundance. The dependency of the apparent rate of CO rebinding on the intensity of the probe beam in single-wavelength experiments is explained.
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