No abstract

No abstract

A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-coxidase catalytic subunits and cytochrome b. Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adeninenucleotide translocator, the opl mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain. After a moderate MnCI, mutagenesis in order to limit multiple mutations, 105 Mit-mutants were isolated from 15000 mutagenised clones in Saccharomyces cerevisiae. Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations). More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, I1 and 111), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein. Mutations in the COXl gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1 , 2 , 3 or exons 5,6,7. Seven missense monosubstitution mutations and two frameshift mutations were also identified. The amino acid changes of the missense mutations were located in the vicinity of the CUB-heme a3 binuclear centre ligands.Cytochrome oxidase, the terminal acceptor of the mitochondrial respiratory chain, catalyses the reduction of oxygen into water. Even if composed of up to 13 subunits, only three subunits I, 11, and ID are mitochondrially coded and appear to be constituents of the minimal catalytic subunits. It is an integral protein complex of the mitochondrial inner membrane which contains four redox-active metal centres: Cu,, hemes a-a3 and CU, (for review, see [l]).The ligands of Cu,, likely to be bound to subunit 11 [2], are not known. Six conserved histidines of subunit I are thought to be the ligands for heme a, heme a3 and Cu,. In bacterial cytochrome oxidase [3] and the corresponding cytochrome o of Escherichia coli [4, 51, the ligands for heme a and the CUB-heme a3 binuclear centre bound to subunit I have been proposed on the basis of site-directed-mutagenesis data. An alternative model of bovine oxidase has also been presented which is still consistent with the site-directed-mutagenesis data and in which the third proposed Cu, ligand and that of the heme a3 are exchanged [6].In order to obtain further insight into the core of the cytochrome c oxidase catalytic centre, a large number of mutagenized clones has been screened in the model organism,

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COXl of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc, complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain.'helve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Cytochrome-c reductase was isolated from Succhuromyces cerevisiue GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDSIPAGE.Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Qo center (Q.ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the K,,, by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The K , of the C137R mutant was not affected and the V,,, was 50% higher.Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDSjPAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Qo center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.All mitochondrial ubiquinol : cytochrome-c reductases comprise three proteins carrying prosthetic groups. These are cytochrome b, the iron-sulfur protein and cytochrome el. Depending on the species, h -8 additional subunits are present. In yeast, the primary sequence of nine subunits has been determined by cloning and sequencing the respective genes [I -81. Sequence alignments showed that all subunits of the yeast enzyme possess homologous counterparts in bovine heart, but the latter contains two additional polypeptides that have so far not been detected in yeast [9]. The function of the non-redox subunits was analyzed by studying the effect of deletion of the particular gene [6. 8, 101. Most of them seem lo be involved in the assembly of mitochondrial cytochromec reductase [I 11.As has been known for a long time, cytochrome h is the only subunit of mitochondrial origin, but deletion of the gene C.'orrespondence to C. von Jagow. Universitatsklinikum, Institut fur Therapeutische Biochemic, lheodor-Stern-Kai 7 Haus 25B, W-6000 Frankfurt 70, Fcderal Rcpublic of Germany Ahhreviutions. NBH, nonylbenzohydroquinone; Q, ubiquinone; TCso, mcdian inhibitory concentration. also causes defects in thc iron-sulfur protein, and the 14-kDa and 11-kDa subunits [12, 131. A different approach to elucidate the structure and function of cytochro...