Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc1 complex of yeast mitochondria

Victor H. Obungu, Yudong Wang, Suzelle M. Amyot, Christian B. Gocke, Diana S. Beattie

Research output: Contribution to journalArticle

Abstract

Resolution of the crystal structure of the mitochondrial cytochrome bc1 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 bc1 complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants ΔVLA(88-90) and ΔAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30°C, enzymatic activities of the bc1 complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, ΔVLA(88-90) and ΔAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c1, 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 ΔVLA(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°C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc1 complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30°C or 37°C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc1 complex.

Original languageEnglish (US)
Pages (from-to)36-44
Number of pages9
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1457
Issue number1-2
DOIs
StatePublished - Feb 24 2000
Externally publishedYes

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Keywords

  • Cytochrome bc
  • Iron-sulphur protein
  • Mitochondria
  • Yeast

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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