Subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein.
Deletion of QCR9, the nuclear gene encoding the 7.3-kDa subunit 9 of the cytochrome bc1 complex, impairs respiration of Saccharomyces cerevisiae, coincident with loss of ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b. Synthesis of cytochrome b by mitochondria from the deletion strain is unimpaired, indicating that the diminished b absorption is due to a post-assembly effect on the heme environment resulting from the absence of subunit 9. Iron-sulfur protein is present in normal amounts and processed to its mature form in the absence of subunit 9, although the protein is more labile to endogenous proteases during the isolation of membranes. EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present. Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in the subunit 9 deletion strain, which suggests that an EPR-silent, sluggishly reactive derivative of the iron-sulfur cluster is present. These results suggest that in the absence of subunit 9 the conformation of iron-sulfur protein is altered such that the protein is more labile, the iron-sulfur cluster is not properly inserted, and iron-sulfur protein interaction with cytochrome b is modified in a manner which distorts the heme environment. This is the first instance in which deletion of one of the supernumerary subunits of the cytochrome bc1 complex results in the loss of function of a redox center within the complex, without a concomitant loss of other subunits.
Phillips
J D
JD
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755.
Graham
L A
LA
Trumpower
B L
BL
D015153:Blotting, Western; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D010101:Oxygen Consumption; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013455:Sulfur / Q000032:analysis
D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D013455:Sulfur; D003575:Cytochromes c1; D008715:Methionine; D007501:Iron; D014450:Electron Transport Complex III