Abstract
The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions. The kinetics of the reaction were examined by varying conditions of ionic strength, inhibitor binding, and oxidation-reduction potential. An analogue of cytochrome c in which the iron atom was replaced with cobalt was used to probe the effect of redox potential on the reaction. Under conditions of low ionic strength, there is very rapid oxidation of cytochrome c and reduction of oxidase which occurs at a rate of 3 X 10(7) M-1 s-1. The number of electrons transferred exhibit a hyperbolic dependence on the concentration of cytochrome c reaching a maximum of 2 electrons transferred at the highest concentration of reduced cytochrome c employed. The total number of electrons transferred was always observed to be distributed equally between cytochrome a and a second acceptor which appears to be the associated copper center; electron transfer to cytochrome a3 did not occur in the absence of oxygen. Substitution of cytochrome c by the cobalt analogue (which represents a decrease in oxidation-reduction potential of about 400 mV) yielded identical results indicating that the origin of the lack of reactivity of cytochrome a3 is of a kinetic nature. The effect of increasing the ionic strength on the reaction was 2-fold: a marked decrease in reaction rate and the appearance of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80% to 40% of the total anticipated from static absorbance measurements. Each of the two phases accounted for a maximum of 1 electron at the highest ionic strength employed. These results are simulated in terms of a sample kinetic reaction scheme involving a two-step electron transfer at one binding site.
Highlights
The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions
The effect of in- in both rate and amplitude andbeheans variouslyinterpreted creasing theionic strength on the reaction was 2-fold: as arisingfrom 1) showdissociation of cytochrome c from a markeddecrease in reaction rate and the appearance cytochrome oxidase [6], 2) conformational changes occurring of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80%to 40% of the total anticipated from static absorbance measurements
Cytochrome a that the rate of reaction between reduced cytochrome c and has been demonstrated tobe an early acceptoorf electrons in cytochrome oxidase decreases with increasing ionic strength cytochrome oxidase through thefollowing observations: 1)the [5, 14], indicating that the interaction between the two proinitial rate of electron transfer due to the oxidation of ferro- teins is electrostatic in nature
Summary
Cytochrome oxidase is isolated frombeef heart essentially as described by Hartzell and Beinert [16] and contains 12-14 nmol of heme a/mg of protein. Cytochrome c was ionic strength is reduced we maintained this procedure for analyzing dissolved in 50 mM Hepes,' pH 7.4, 0.5%Tween 20, 15 m~ EDTA to the data obtained at low ionic strength. Under the latter the desired concentration using an extinction coefficient of 11.0 rn"'. Of 29 m"' cm" at 550 nm for the reduced protein [22].Cytochrome The absorbance changes observed at each wavelength in each oxidase was found to be much less susceptible to photoreduction by exponential phase of the reaction are corrected for the relative conthis method and under these conditions only partial reduction of tributions of cytochromes a and a3and cytochrome c.
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