cytochromes-c1 and 2-3-dimethoxy-5-methyl-6-decyl-1-4-benzoquinone

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.

Topics: Amino Acid Sequence; Binding Sites; Cell Respiration; Conserved Sequence; Cytochromes b; Cytochromes c1; Electron Transport; Glutamic Acid; Hydrogen-Ion Concentration; Hydroquinones; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes; Mutagenesis, Site-Directed; Mutant Proteins; Oxidation-Reduction; Protein Binding; Protons; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Ubiquinone

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six "human" mutations, namely G33S, S152P, G252D, Y279C, G291D, and Delta252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and Delta252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc(1) complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc(1) complex. Y279C caused a significant decrease of the bc(1) function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Q(o) site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Q(o) site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.

Topics: Aspartic Acid; Binding Sites; Blotting, Western; Cytochromes b; Cytochromes c; Cytochromes c1; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Fungal Proteins; Genetic Diseases, Inborn; Humans; Immunoblotting; Intracellular Membranes; Iron-Sulfur Proteins; Kinetics; Lipids; Magnetics; Mitochondria; Models, Molecular; Mutation; Saccharomyces cerevisiae Proteins; Spectrophotometry; Suppression, Genetic; Temperature; Ubiquinone