Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.     

Electron transfer and stability of the cytochrome b6f complex in a small domain deletion mutant of cytochrome f

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.     

Disruption of the interaction between the Rieske iron-sulfur protein and cytochrome b in the yeast bc1 complex owing to a human disease-associated mutation within cytochrome b.

The mitochondrial cytochrome b missense mutation, G167E, has been reported in a patient with cardiomyopathy. The residue G167 is located in an extramembranous helix close to the hinge region of the iron-sulfur protein. In order to characterize the effects of the mutation on the structure and function of the bc(1) complex, we introduced G167E into the highly similar yeast cytochrome b. The mutation had a severe effect on the respiratory function, with the activity of the bc(1) complex decreased to a few per cent of the wild type. Analysis of the enzyme activity indicated that the mutation affected its stability, which could be the result of an altered binding of the iron-sulfur protein on the complex. G167E had no major effect on the interaction between the iron-sulfur protein headgroup and the quinol oxidation site, as judged by the electron paramagnetic resonance signal, and only a minor effect on the rate of cytochrome b reduction, but it severely reduced the rate of cytochrome c(1) reduction. This suggested that the mutation G167E could hinder the movement of the iron-sulfur protein, probably by distorting the structure of the hinge region. The function of bc(1) was partially restored by mutations (W164L and W166L) located close to the primary change, which reduced the steric hindrance caused by G167E. Taken together, these observations suggest that the protein-protein interaction between the n-sulfur protein hinge region and the cytochrome b extramembranous cd2 helix is important for maintaining the structure of the hinge region and, by consequence, the movement of the headgroup and the integrity of the enzyme.     

Mitochondrial encephalomyopathy and complex III deficiency associated with a stop-codon mutation in the cytochrome b gene

We have reinvestigated a young woman, originally reported by us in 1983, who presented with exercise intolerance and lactic acidosis associated with severe deficiency of complex III and who responded to therapy with menadione and ascorbate. Gradually, she developed symptoms of a mitochondrial encephalomyopathy. Immunocytochemistry of serial sections of muscle showed a mosaic of fibers that reacted poorly with antibodies to subunits of complex III but reacted normally with antibodies to subunits of complexes I, II, or IV, suggesting a mutation of mtDNA. These findings demonstrate the diagnostic value of immunocytochemistry in identifying specific respiratory-chain deficiencies and, potentially, distinguishing between nuclear- or mtDNA-encoded defects. Sequence analysis revealed a stop-codon mutation (G15242A) in the mtDNA-encoded cytochrome b gene, resulting in loss of the last 215 amino acids of cytochrome b. PCR-RFLP analysis indicated that the G15242A mutation was heteroplasmic and was present in a high percentage (87%) of affected tissue (skeletal muscle) and a low percentage (0.7%) of unaffected tissue (blood) but was not detected in controls. Analysis of microdissected muscle fibers showed a significant correlation between the immunoreactivity toward the Rieske protein of complex III and the percentage of mutant mtDNA: immunopositive fibers had a median value of 33% of the G15242A mutation, whereas immunonegative, ragged-red fibers had a median value of 89%, indicating that the stop-codon mutation was pathogenic in this patient. The G15242A mutation was also present in several other tissues, including hair roots, indicating that it must have arisen either very early in embryogenesis, before separation of the primary germ layers, or in the maternal germ line. The findings in this patient are contrasted with other recently described patients who have mutations in the cytochrome b gene.     

Molecular analysis of revertants from a respiratory-deficient mutant affecting the center o domain of cytochrome b in Saccharomyces cerevisiae

In bc complexes, cytochrome b plays a major role in electron transfer and in proton translocation across the membrane. Several inhibitor-resistant and respiratory-deficient mutants have already been used to study the structure-function relationships of this integral membrane protein. We describe here the selection and the molecular analysis of revertants from a thermo-sensitive mit-mutant of known nucleotide changes. Among 80 independent pseudo-wild type revertants screened by DNA-labelled oligonucleotide hybridization, 33 have been sequenced. Eight suppressor mutations, affecting a region critical for both the function and the binding of center o inhibitors (end of helix C) were identified. Two of them were found to be more resistant to myxothiazol.     

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a3 2+ . CO.

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity. Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a3 in the mixed-valence enzyme after photodissociation. Upon addition of CO to partially reduced formate cytochrome c oxidase (a2+a3 3+ . HCOOH) the cytochrome a3 2+. CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a3 and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a3 3+ . HCOOH compound is dissociated slowly with a concomitant formation of the a3 2+ . CO compound and oxidation of cytochrome a. When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a3 and the a2+ 3 . CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

Nucleotide sequence of the gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex.

The nucleotide sequence was determined for the first part of the Bacillus subtilis sdh operon. An open reading frame corresponding to the structural gene, sdhA, for cytochrome b558 was identified. The predicted molecular weight of the cytochrome (excluding the N-terminal methionine) is 22,770. It is a very hydrophobic protein with five probable membrane-spanning segments. There is little homology between the B. subtilis cytochrome b558 and cytochrome b of mitochondrial complex III from different organisms or between cytochrome b558 and the hydrophobic sdhC and sdhD peptides of the Escherichia coli sdh operon. About 30 bases downstream of the sdhA stop codon, a new open reading frame starts. The nucleotide sequence predicts the presence of a typical flavin-binding peptide which identifies this reading frame as part of the sdhB gene. Seven bases upstream of the sdhA initiation codon ATG there is a typical B. subtilis ribosome binding site (free energy of interaction, -63 kJ), and further upstream, tentative sigma 55 and sigma 32 promoter sequences were found. The upstream region also contains two 12-base-long direct repeats; their significance is unknown.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene

Summary Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci. In all cases so far, the site of Ty insertion has been in the 5 non-coding region of the affected gene. Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene. This Ty insertion within a gene has a similar structure to those in non-coding regions.     

The manipulation of cellular cytochrome and lipid composition in a haem mutant of Saccharomyces cerevisiae.

1. The ole-3 mutant of Saccharomyces cerevisiae has an early lesion in the pathway of porphyrin biosynthesis. 2. This results in the loss of all haem-containing enzymes, including the mitochondrial cytochromes, and prevents the synthesis of components whose formation requires haem-containing enzymes, including unsaturated fatty acids, ergosterol and methionine. 3. The pleiotropic effects of the primary lesion are reversed by growing mutant ole-3 aerobically in the presence of intermediates of the porphyrin-biosynthetic pathway, and the present work reports the degree of manipulation of lipid and respiratory-cytochrome composition. 4. Supplements of delta-aminolaevulinate in the range 0.5--500 mg/l result in a progressive increase in the cellular content of unsaturated fatty acids and respiratory cytochromes, cause the replacement of lanosterol and squalene by ergosterol, and an increase in total sterol content. 5. Haematoporphyrin and protoporphyrin IX have similar but less extensive effects on cellular composition, whereas haematin allows unsaturated fatty acid synthesis and some sterol synthesis, but has no effect on the formation of respiratory cytochromes. 6. These results suggest that growth of the organism in the presence of defined amounts of delta-aminolaevulinate will be useful in the investigation of the role of lipids and cytochromes in the function and assembly of mitochondrial membranes.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Structure of a cytochrome b-c 1 complex from Saccharomyces cerevisiae YF.

1) An isolation and purification procedure is reported for an active cytochrome b-c1 complex from Saccharomyces cerevisiae. The complex acts as an antimycin A-sensitive duroquinone-cytochrome c reductase and contains cytochromes b and c1 at a concentration of 8 nmol/mg protein and non-heme iron at a concentration of 15 nmol/mg protein. 2) Difference spectra at room temperature and at 70 degrees K show that the preparation is free from contamination with cytochromes c or aa3. Assays of enzyme activity indicate the absence of any of the other catalytic functions normally associated with the mitochondrial respiratory chain. 3) On dissociation and separation on sodium dodecylsulfate-polyacrylamide gels the complex gives rise to seven bands corresponding to subunit polypeptide molecular weights of 43 000, 40 000, 32 000, 24 000, 22 000, 20 000 and 18 000. These appear in a regular stoichiometry of 1:1:3:1:1:1:1.