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.
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.
Ghosh
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M
Department of Biochemistry, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9142, USA.
Wang
Y
Y
Ebert
C E
CE
Vadlamuri
S
S
Beattie
D S
DS
D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000235:genetics; D003198:Computer Simulation; D017124:Conserved Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D003575:Cytochromes c1 / Q000737:chemistry / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009245:NADH Dehydrogenase / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D011090:Polyenes / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D000900:Anti-Bacterial Agents; D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D007506:Iron-Sulfur Proteins; D010446:Peptide Fragments; D011090:Polyenes; D003575:Cytochromes c1; C041573:stigmatellin; D009245:NADH Dehydrogenase; D014450:Electron Transport Complex III; D007930:Leucine; D000409:Alanine