ANALYSIS OF CYTOCHROME-B AMINO-ACID-RESIDUES FORMING THE CONTACT FACEWITH THE IRON-SULFUR SUBUNIT OF UBIQUINOL - CYTOCHROME-C REDUCTASE INSACCHAROMYCES-CEREVISIAE

Four mutations in the mitochondrial cytochrome b of Saccharomyces cere visiae have been characterized with respect to catalytic properties, i nhibitor resistance and subunit interaction. The respiratory-deficient mutant [G137E]cytochrome b and the pseudo-wild-type revertant [G137E, N256K]cytochrome b were described previously [di Rage, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332-3339; di Rage, J.-P., Netter, P. and Slonimski, P. P, (1990) J. Biol. Chem. 265, 1575 0-15757]. Two new mutants [N256K]cytochrome b and [N256K]cytochrome b were isolated by dissociation of the second-site suppressor from the o riginal target mutation. The mutants [G137E]cytochrome b and [G137E, N 256K]cytochrome b exhibited a high resistance against methoxyacrylate inhibitors, whereas the suppressors [N256K]cytochrome b and [N256I]cyt ochrome b showed only a slight resistance. Remarkably, all mutants exh ibited stigmatellin cross-resistance. The electron-transfer activity f rom the substrate nonylubiquinol to cytochrome c of mitochondrial memb ranes was diminished in all mutants. The substitution G137-->E decreas es V,,IK, by one order of magnitude, indicating a reduced catalytic ef ficiency for ubiquinol. The amino acid exchange at position 256 to a p ositively charged lysine residue or to a hydrophobic isoleucine residu e resulted mainly in a diminished specific activity. The iron-sulfur s ubunit and the 8.5-kDa subunit were detectable in all mutants at norma l levels in immunoblots of membrane preparations, indicating proper as sembly of the complex. However, after purification, the mutant be, com plex lacked the iron-sulfur subunit and the: 8.5-kDa subunit. In contr ast, the iron-sulfur subunit can only be dissociated from the parental be, complex by harsh treatment. These data suggest that residues 137 and 256 in cytochrome b are crucial for cytochrome-bl iron-sulfur prot ein interaction.