Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol--cytochrome-c reductase by the binding of ubiquinone.
1. The quenching by ubiquinone (Q) of the intrinsic fluorescence of tryptophan residues within ubiquinol--cytochrome-c reductase (complex III) has been exploited to provide direct information on the interaction between these two components of the mitochondrial respiratory chain. 2. The fluorescence quenching data have been corrected for inner filter effects and interpreted using the classical Stern-Volmer and modified Stern-Volmer plots. The latter of these plots allows computation of both the dissociation constant (Kd) of complex formation between ubiquinone and complex III, and the percentage of fluorophores accessible to quenching. 3. It is found that different Q homologues bind to complex III with different affinities depending upon the length of the isoprenoid chain: 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone, an analogue of Q2, exhibits the same Kd as Q2. Furthermore, the accessibility of fluorophores to quenching was lower for Q1 than for the other quinones tested. 4. The binding affinity of Q2 to complex III depends upon the redox state of the enzyme. 5. Addition of the complex III inhibitor, antimycin, has very little effect on the binding affinity or on the accessibility of fluorophores to the quencher. 6. Addition of the inhibitor myxothiazol has a similar effect to reducing complex III with ascorbate. 7. Reconstitution of complex III into asolectin lipid vesicles gives similar qualitative results to the enzyme in solution regarding both the redox state and the addition of inhibitors.
Samworth
C M
CM
Department of Biology, University of Bologna, Italy.
Degli Esposti
M
M
Lenaz
G
G
D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan; D014451:Ubiquinone / Q000378:metabolism*
D003573:Cytochrome b Group; D014451:Ubiquinone; D014364:Tryptophan; D003575:Cytochromes c1; D014450:Electron Transport Complex III