Direct demonstration of half-of-the-sites reactivity in the dimeric cytochrome bc1 complex: enzyme with one inactive monomer is fully active but unable to activate the second ubiquinol oxidation site in response to ligand binding at the ubiquinone reduction site.
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.
Castellani
Michela
M
Institute of Biochemistry, Molecular Genetics, Goethe University and Cluster of Excellence Macromolecular Complexes Frankfurt am Main, D-60438 Frankfurt am Main, Germany.
Covian
Raul
R
Kleinschroth
Thomas
T
Anderka
Oliver
O
Ludwig
Bernd
B
Trumpower
Bernard L
BL
D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D002846:Chromatography, Affinity; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006736:Horses; D007700:Kinetics; D008024:Ligands; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction / Q000187:drug effects; D010231:Paracoccus denitrificans / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects; D017186:Titrimetry; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism*
D008024:Ligands; D050505:Mutant Proteins; C032456:antimycin; D014451:Ubiquinone; D000968:Antimycin A; D014450:Electron Transport Complex III; C003741:ubiquinol