scholarly journals Protonmotive functions of cytochrome c oxidase in reconstituted vesicles. Influence of turnover rate on ‘proton translocation’

1983 ◽  
Vol 210 (1) ◽  
pp. 199-205 ◽  
Author(s):  
G Proteau ◽  
J M Wrigglesworth ◽  
P Nicholls
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Turnover Rate ◽  
External Medium ◽  
Proton Translocation ◽  
Buffering Capacity ◽  
Ph Gradient ◽  
Ferrocytochrome C ◽  
Carbonyl Cyanide ◽  
Enzyme Turnover

1. Oxidation of ferrocytochrome c by cytochrome c oxidase incorporated into proteoliposomes induces a transient acidification of the external medium. This change is dependent on the presence of valinomycin and can be abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone or by nigericin. The H+/e- ratio for the initial acidification varies with the internal buffering capacity of the vesicles, and under suitable conditions approaches + 1, the pulse slowly decaying to give a net alkalinity change with H+/e- value approaching −1. 2. Inhibition of cytochrome c oxidase turnover by ferricytochrome c or by azide addition results in ferrocytochrome c-dependent H+ pulses with decreasing H+/e- ratios. The rate of the initial H+ production remains higher than the rate of equilibration of the pH gradient, indicating an intrinsic dependence of the H+/e- ratio on enzyme turnover. The final net alkalinity changes are relatively unaffected by turnover inhibition.

scholarly journals Limited-turnover studies on proton translocation in reconstituted cytochrome c oxidase-containing vesicles

1979 ◽  
Vol 182 (1) ◽  
pp. 149-156 ◽  
Author(s):  
R P Casey ◽  
J B Chappell ◽  
A Azzi
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Proton Pump ◽  
Proton Translocation ◽  
Strong Support ◽  
Ferrocytochrome C ◽  
Vesicle Membranes ◽  
Careful Control ◽  
Hydrogen Carrier ◽  
Reconstituted System

We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

1977 ◽  
Vol 55 (10) ◽  
pp. 1114-1117 ◽  
Author(s):  
Gerrit H. Bomhoff ◽  
Mary Spencer
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Ferrocytochrome C ◽  
Optimum Ph ◽  
Triton X ◽  
Assay Conditions ◽  
Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

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