Abstract
The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.
Highlights
The mitochondrial respiratory chain couples electron transfer to proton translocation across the inner membrane, thereby maintaining a proton electrochemical gradient that drives transmembrane transport as well as formation of ATP
We investigated ligand binding to heme a3 and internal electron transfer between hemes a and a3 as a function of the reduction pressure on the purified cytochrome c oxidase (CytcO)
Upon reduction of heme a3, another CO molecule binds to the reduced heme a3 thereby increasing its apparent midpoint potential[12]
Summary
The mitochondrial respiratory chain couples electron transfer to proton translocation across the inner membrane, thereby maintaining a proton electrochemical gradient that drives transmembrane transport as well as formation of ATP. Genetic removal of the protein results in a decrease in the O2-reduction activity to ~30% of that observed for CytcO in wild-type mitochondria[4,9], presumably due to structural changes in a fraction of the CytcO population[10,11]. These structural changes are most noticeably reflected in changes in the kinetics of CO-ligand binding, which is accelerated by a factor of ~102. O2, results in gradual reduction of the enzyme In this redox reaction CO is oxidized to CO2 while two electrons are transferred to CytcO. The extent of the inter-heme electron transfer varies between CytcOs from different species depending on the relative midpoint potentials of hemes a and a3
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