Modification of the Spectral Properties of Cytochrome b in Mutants of Saccharomyces cerevisiae Resistant to 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea

Abstract

The effects of five inhibitors of the cytochrome bc1 complex: 3‐(3.4‐dichlorophenyl)‐1,1‐dimethylurea (diuron), 2‐n‐heptyl‐4‐hydroxyqttinoline‐N‐oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuronresistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. The oxidation of NADH and of cytochronic b‐561 exhibits a similar resistance to diuron and HpHOQnO in Diu I and Diu2 mutants. No extra reduction of cytochrome b‐561 and cytochrome b‐565 is observed in the presence of diuron and HpHOQnO. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b‐561 induced by concentrations of HpHOQnO 2‐3‐times higher than those required in the parental strain. The spectral and respiratory effects of antimycin A, funiculosin and mucidin are generally similar in the diuron‐resistant mutants and in their parental strains. However a cross‐resistance between diuron and antimycin A is indicated in one Diu 2 mutant. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in tile central region of tile apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b‐565 and b‐561 as well as tile flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b‐561 is only slightly affected by the diu‐l and diu‐2 mutations.

Mitochondrial cytochrome b: evolution and structure of the protein

“…The problem is to correlate changes In the tltre of one inhibitor with one (or few) specific residue substitutions that naturally occur in the sequence of cytochrome b This problem is complicated by the absence of a known three-dimensional structure of the protein and by the difficulty of finding a strict one-toone relationship Two considerations, however, mitigate the problems (1) The detailed knowledge of the available resistant mutants can be used as a guide for locating the protein regions or deducing the type of amino acid replacement that may cause a gwen inhibitor response [9,19,26,39,73,90,129,134,137] (2) The natural variants of a protein are stable and fully functional [75,140] In contrast, mutated proteins generally have major functional derangements [59,72,75] (see [8,18,68,76,133,135,137,141] for cytochrome b mutants) Moreover, the 'element of surprise' [140] in the natural amino acid variation can prowde multiple substitutions for assessing the role of specific residues…”

“…Functionally, the center i inhlbitors block the reoxadatlon of cytochrome b and destabilize the bound ubisemiquinone [8,9,80,106,125] Antlmycin is the most powerful of these compounds (see Ref 113 for a review), but it is by no means a unwersal inhibitor This antibiotic, in fact, is not potent in the bc 1 complex of parasitic nematodes [127] and of the protozoan Tetrahyrnena [39], and quite ineffective in chloroplast bf complexes [3,121] Table III lists all known mutations affecting the sensitivity towards center i lnhIbltors in mltochondnal cytochrome b The mutated residues consistently lie within transmembrane helices A, D and E and lead to an Increase in the volume of the exchanged residue (Fig 2 and Table III, cf Refs 6,8,12,90,[128][129][130][131][132][133][134][135][136] With the exception of the mouse mutant G232 ~ D [135], the mutatmns do not slgmficantly alter the turnover of the reductase and, in general, produce a hmlted increase m the tltre of the mhlbltors (Table III and [26,[128][129][130][131][132][133][134][135][136]) These p r o p e m e s of the mutations leading to resistance towards center i lnh~bltors are slmdar to those exhibited by the herblode-reslstant mutations that map w~thIn the transmembrane hehces of the photosynthetic subumts [116]…”

“…Can we exploit the natural variation of cytochrome b to d e t e r m m e structure-function relationships 9 Some speculations on naturally occurring sites of altered sensltwlty towards bc 1 lnhlbltors have already been made [6,19,26,39,46,90,[129][130][131][132][133][134][135][136][137] In order to substantiate speculations of this kind, a thorough investigation of the sequence to property relat~onship must be undertaken in several variants of a protein [72,140] The numerous sequences of cytochrome b that are available and the posslblhty of measuring inhibitor binding from the tltratlons of the cytochrome c reductase actwity in mitochondrla [36,39,46,90,115,[133][134][135][136]138] have allowed us to systematically carry out this investigation Natural resistance has been found in many species (Table V), thus prowdmg cases of altered properties that could be related to structural changes m the natural variants of cytochrome b…”