Abstract
Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.
Highlights
+ + + the heme is reduced by an equivalent of the water-gas shift reaction (CO HzO + COz 2e- 2H’)
We propose that autoreduction of cytochrome c oxidase is the reduction of copper and ferricheme iron,“driven” by carbon monoxide oxidation: and dissociation of the peroxide adduct
In the absence of oxygen, if a mixture of cytochromec and cytochrome c oxidase is incubated under a CO atmosphere, autoreduction of the cytochrome c as well as of the cytochrome c oxidase occurs
Summary
HbAw' as prepared by the ammonium sulfate procedure and stripped of ions as described previously [7].Oxidized metHbA was prepared by treating oxy-HbA in 0.1 M NaHP04, pH7.4,with NaN02 and dialyzing against three changes of 500 volumes of the same buffer. The amount of base added was measured, and the acid equivalent produced during reduction was determined This procedure avoided possible errors associated with the buffering capacity of the buffer and protein solution. The specrates of CO-driven reduction by different proteins were done in sealed trum of the reduced and partially reduced species resemble cuvettes, thoroughly degassed and saturated with CO in tandem.High pressure experiments with metHbA were performed at 20 "C in a Parr Bomb (Parr Instruments Co.) that was bubbled with argon for 5 min, subjected to 1500psi of CO, and sampled at regular intervals. No heme reduction was detectable after 200 or more hours for solutions of catalase, cytochrome c (alone), or horseradish peroxidase, proteins that cannotbind CO even when reduced. Protein anddipyridine- or &nicotinic acid-liganded heme solutions dependence of the CO recombination after flashphotolysis at were in 0.1 M KHPO,, pH 7.5, 0.1 mM EDTA a t 25 “ C .Samples of cytochrome c oxidase contained 1% Tween 80.
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