Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae.
Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.
Brasseur
G
G
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, Marseilles, France. brasseur@ibsm.cnrs-mrs.fr
Di Rago
J P
JP
Slonimski
P P
PP
Lemesle-Meunier
D
D
D001665:Binding Sites; D002384:Catalysis; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D003575:Cytochromes c1 / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011199:Potentiometry; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013053:Spectrophotometry; D013489:Suppression, Genetic
D003573:Cytochrome b Group; D003575:Cytochromes c1; D010088:Oxidoreductases; C022691:succinate oxidase; D014450:Electron Transport Complex III