title: Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria.
abstract: Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.
authors.affiliation: Department of Biochemistry, P.O. Box 9142, West Virginia University School of Medicine, Morgantown, WV 26506-9142, USA.
authors.forename: V H
authors.initials: VH
authors.lastname: Obungu
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: S M
authors.initials: SM
authors.lastname: Amyot
authors.forename: C B
authors.initials: CB
authors.lastname: Gocke
authors.forename: D S
authors.initials: DS
authors.lastname: Beattie
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology*; D016254:Mutagenesis, Insertional; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D007506:Iron-Sulfur Proteins; D014450:Electron Transport Complex III