The “Ferrous-Oxy” Intermediate in the Reaction of Dioxygen with Fully Reduced Cytochromesaa3andbo3

Abstract
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 α-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 μs; 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 μs and has a broad maximum near 570 nm.

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