Abstract
Abstract The losses of molecular activity which take place when cytochrome oxidase is purified from bovine heart muscle particles by the procedures of Yonetani ((1960) J. Biol. Chem. 235, 845) and of Fowler et al. ((1962) Biochim. Biophys. Acta 64, 170) have been examined. Bovine heart muscle particles (about 1 nmole of heme a per mg of protein) and oxidase-rich particles (about 4 nmoles of heme a per mg of protein) treated with deoxycholate under optimal conditions, had molecular activities (MA0,max) (at infinitely high concentration of cytochrome c, spectrophotometrically determined in 0.05 m phosphate buffer, pH 7.0, 25°) in the range of 530 to 580 s-1. MA0,max, determined under identical reaction conditions, declined with successive steps in the Yonetani and Fowler et al. purification procedures for cytochrome oxidase, to considerably lower values for the finally isolated enzymes, while Kmapp stayed relatively unchanged. Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, showed no evidence of essential component removal during purification, that might account for decreased MA0,max values. The accumulation of an endogenous inhibitor in the course of preparation was also shown to be an unlikely cause for the observed decline of MA0,max. Treatment with limited quantities of dodecyl sulfate led to stimulation of activity of Yonetani's preparation. Similar treatment of Fowler's preparation and of deoxycholate-treated, oxidase-rich particles resulted only in decreased molecular activity. Dodecyl sulfate appeared to be an uncompetitive inhibitor of cytochrome oxidase. The aerobic ferrocytochrome c oxidation catalyzed by deoxycholate-solubilized particulate oxidase exhibited apparent first order kinetics over at least 99% of the reaction course. Purified oxidase preparations showed deviations from first order (concave semilogarithmic reaction plots) at low remaining substrate concentrations. This could not be ascribed either to spontaneous activation in the reaction mixture or to unequal affinities of the oxidase for ferrous and ferric forms of cytochrome c. Reversible unproductive binding of ferrocytochrome c with a fraction of the oxidase in the purified preparations explains this. Deoxycholate-treated oxidase particles exhibited a largely oxidized 605 nm band during the aerobic steady state in the system containing ascorbate, cytochrome c, oxidase, and O2, whereas Yonetani's enzyme appeared appreciably reduced. This indicates a blockade of the electron transfer from a to a3 in the inactive or latent portion of the oxidase preparation. It appears from these observations that some portion of the original membrane-bound cytochrome oxidase may be transformed during purification into catalytically inactive or latent species, possibly by reorganization of the original multimeric phospholipid-protein complex in a portion of the purified enzymes. Various physical, chemical, and catalytic complexities which have been observed with chemically purified cytochrome oxidases may have their origins in molecular heterogeneities.
Highlights
The losses of molecular activity which take place when cytochrome oxidase is purified from bovine heart muscle particles by the procedures of Yonetani ((1960) J
Yonetani’s enzyme appeared appreciably reduced. This indicates a blockade of the electron transfer from a to a3 in the inactive or latent portion of the oxidase preparation. It appears from these observations that some portion of the original membrane-bound cytochrome oxidase may be transformed during purification into catalytically inactive or latent species, possibly by reorganization of the original multimeric phospholipid-protein complex in a portion of the purified enzymes
A number of observations suggest that structure and function is maintained during extraction and purification: (a) similar reaction order and dependence of the rate constant on the sum of the concentrations of ferrous and ferric cytochrome c of exogenous-cytochrome c oxidation catalyzed by oxidase in situ [3,4,5,6] and of enzyme detached from membrane [7] and further purified [6, 8,9,10,11,12] ; (b) sensitivity of purified cytochrome oxidase to respiratory inhibitors such as cyanide (e.g. Ref. 13), azide (e.g. Ref. 14) and carbon monoxide [15] ; (c) similar effects produced by changes
Summary
The losses of molecular activity which take place when cytochrome oxidase is purified from bovine heart muscle particles by the procedures of Yonetani ((1960) J. This indicates a blockade of the electron transfer from a to a3 in the inactive or latent portion of the oxidase preparation It appears from these observations that some portion of the original membrane-bound cytochrome oxidase may be transformed during purification into catalytically inactive or latent species, possibly by reorganization of the original multimeric phospholipid-protein complex in a portion of the purified enzymes. A number of observations suggest that structure and function is maintained during extraction and purification: (a) similar reaction order and dependence of the rate constant on the sum of the concentrations of ferrous and ferric cytochrome c of exogenous-cytochrome c oxidation catalyzed by oxidase in situ [3,4,5,6] and of enzyme detached from membrane [7] and further purified [6, 8,9,10,11,12] ; (b) sensitivity of purified cytochrome oxidase to respiratory inhibitors such as cyanide (e.g. Ref. 13), azide (e.g. Ref. 14) and carbon monoxide [15] ; (c) similar effects produced by changes. We discuss the nature of the changes which result in the loss of molecular activity in purified oxidase preparations
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