The "ferrous-oxy" intermediate in the reaction of dioxygen with fully reduced cytochromes aa3 and bo3.

We have studied the reactions with oxygen of two terminal oxidases, cytochrome c oxidase from mitochondria and cytochrome bo3 from Escherichia coli. In each case, flow-flash methodology was used to react the fully reduced enzyme with a high concentration of oxygen (1 mM), and absorbance changes were recorded for a number of separate wavelengths in the alpha-band (visible) region. In both enzymes, an early kinetic phase could be resolved, corresponding to the binding of oxygen to produce a ferrous-oxy heme intermediate. In cytochrome c oxidase, this intermediate appears with a time constant of 10 microseconds; its spectrum has a peak at 595 nm (relative to the unliganded reduced enzyme). In cytochrome bo3, the ferrous-oxy intermediate, resolved by optical absorbance spectroscopy for the first time, appears with a time constant of 11 microseconds and has a broad maximum near 570 nm.