Substituting Leucine for Alanine-86 in the Tether Region of the Iron−Sulfur Protein of the Cytochrome bc1 Complex Affects the Mobility of the [2Fe2S] Domain

• Published in: Biochemistry (Easton) 2001-01, Vol.40 (2), p.327-335
• Main Authors: GHOSH, Mousumi, WANG, Yudong, EBERT, C. Edward, VADLAMURI, Satya, BEATTIE, Diana S.
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Alanine - genetics; Amino Acid Substitution - genetics; Anti-Bacterial Agents - pharmacology; Biological Transport - genetics; Computer Simulation; Conserved Sequence; Cytochrome b Group - chemistry; Cytochrome b Group - genetics; Cytochromes c1 - chemistry; Cytochromes c1 - genetics; Electron Transport Complex III - chemistry; Electron Transport Complex III - genetics; Electron Transport Complex III - isolation & purification; Enzyme Activation - genetics; Enzyme Inhibitors - pharmacology; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; Kinetics; Leucine - chemistry; Leucine - genetics; Models, Molecular; NADH Dehydrogenase - antagonists & inhibitors; Oxidation-Reduction; Peptide Fragments - chemistry; Peptide Fragments - genetics; Point Mutation; Polyenes - pharmacology; Protein Structure, Tertiary - genetics; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001708t

Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.