Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria

The reaction of mixed valence state cytochrome oxidase (Cu(A) (2+)a(3+) . Cu(B) (+)a(3) (2+)) with O(2) at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The "pure" difference spectra of the two species, I(M) and II(M), relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I(M) is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II(M) by an alpha band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O(2) (-) --> Cu(B) (2+) and O(2) (-) --> a(3) (2+) charge transfer transitions which would imply that O(2) (-) forms a bridging ligand between Cu(B) and the iron atom of cytochrome a(3) in species II(M). The kinetics of the reaction and the spectral characteristics of species I(M) and II(M) obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.