Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc(1) 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% dec reases in enzymatic activity [Obungu et al, (2000) Biochim. Biophys. Acta 1 457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86 L was decreased 60% compared to the wild-type without loss of heme or prote in 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-ty pe, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither muta tion A86L nor mutation A92R affects the alpha -helical backbone in the teth er region; however, the side chain of the leucine substituted for Ala-86 in teracts with the side chain of Leu-89. The Arrhenius plot for mutant A86L w as apparently biphasic with a transition observed at 17-19 degreesC and an activation energy of 279.9 kJ/mol below 17 degreesC and 125.1 kJ/mol above 17 degreesC. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was una ffected, suggesting that movement of the tether region of the iron-sulfur p rotein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha -helix and hence mov ement of the tether.