Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms

2012 ◽  
Vol 444 (2) ◽  
pp. 199-204 ◽  
Author(s):  
Brigitte Meunier ◽  
Amandine Maréchal ◽  
Peter R. Rich
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Point Mutations ◽  
Normal Functioning ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Terminus ◽  
Radical State ◽  
Uv Visible ◽  
H Channels ◽  
Mutant Forms

Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s−1 (electrons per s)], UV–visible signatures of oxidized and reduced states and ability to form the PM [‘peroxy’ (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.

scholarly journals Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4961-4972 ◽  
Author(s):  
Norbert Gattermann ◽  
Stefan Retzlaff ◽  
Yan-Ling Wang ◽  
Götz Hofhaus ◽  
Jürgen Heinisch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Ferric Iron ◽  
Point Mutations ◽  
Polymorphism Analysis ◽  
Sideroblastic Anemia ◽  
Wide Range

Mitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.

scholarly journals Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4961-4972 ◽  
Author(s):  
Norbert Gattermann ◽  
Stefan Retzlaff ◽  
Yan-Ling Wang ◽  
Götz Hofhaus ◽  
Jürgen Heinisch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Ferric Iron ◽  
Point Mutations ◽  
Polymorphism Analysis ◽  
Sideroblastic Anemia ◽  
Wide Range

Abstract Mitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.

scholarly journals Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

2014 ◽  
Vol 2014 ◽  
pp. 1-5
Author(s):  
Verónica Loera-Castañeda ◽  
Lucila Sandoval-Ramírez ◽  
Fermín Paul Pacheco Moisés ◽  
Miguel Ángel Macías-Islas ◽  
Moisés Alejandro Alatorre Jiménez ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Alzheimer Disease ◽  
Cytochrome C Oxidase ◽  
Point Mutations ◽  
Mitochondrial Cytochrome ◽  
Catalytic Core ◽  
Oxidase Gene ◽  
Mexican Patients ◽  
Mitochondrial Cytochrome C

Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients.MTCO IIgene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

scholarly journals The mechanism for the ATP-induced uncoupling of respiration in mitochondria of the yeast Saccharomyces cerevisiae

1995 ◽  
Vol 307 (3) ◽  
pp. 657-661 ◽  
Author(s):  
S Prieto ◽  
F Bouillaud ◽  
E Rial
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Kinetic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Low Efficiency ◽  
Respiratory Chain Activity

We have recently reported that ATP induces an uncoupling pathway in Saccharomyces cerevisiae mitochondria [Prieto, Bouillaud, Ricquier and Rial (1992) Eur. J. Biochem. 208, 487-491]. The presence of this pathway would explain the reported low efficiency of oxidative phosphorylation in S. cerevisiae, and may represent one of the postulated energy-dissipating mechanisms present in these yeasts. In this paper we demonstrate that ATP exerts its action in two steps: first, at low ATP/Pi ratios, it increases the respiratory-chain activity, probably by altering the kinetic properties of cytochrome c oxidase. Second, at higher ATP/Pi ratios, an increase in membrane permeability leads to a collapse in membrane potential. The ATP effect on cytochrome c oxidase corroborates a recent report showing that ATP interacts specifically with yeast cytochrome oxidase, stimulating its activity [Taanman and Capaldi (1993) J. Biol. Chem. 268, 18754-18761].

scholarly journals The Yeast Aac2 Protein Exists in Physical Association with the Cytochromebc1-COX Supercomplex and the TIM23 Machinery

2008 ◽  
Vol 19 (9) ◽  
pp. 3934-3943 ◽  
Author(s):  
Mary K. Dienhart ◽  
Rosemary A. Stuart
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Affinity Purification ◽  
Yeast Saccharomyces Cerevisiae ◽  
Present Evidence ◽  
Cytochrome Bc ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Physical Association

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc1)-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc1-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc1-COX supercomplex is perturbed, whereby a decrease in the III2-IV2assembly state relative to the III2-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc1-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc1-COX-TIM23 supercomplex are also discussed.

scholarly journals Structure/function of oxygen-regulated isoforms in cytochrome c oxidase.

1998 ◽  
Vol 201 (8) ◽  
pp. 1163-1175
Author(s):  
P V Burke ◽  
R O Poyton
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Transmembrane Helix ◽  
Multigene Families ◽  
Low Oxygen ◽  
Developmentally Regulated ◽  
Yeast Saccharomyces Cerevisiae ◽  
Catalytic Constant ◽  
Different Types

Eukaryotic cytochrome c oxidases are complex oligomeric membrane proteins composed of subunit polypeptides encoded by both nuclear and mitochondrial genomes. While the mitochondrially encoded subunits are encoded by unique genes, some of the nuclear-encoded subunits are encoded by multigene families. The isoforms produced by these multigene families are tissue-specific and/or developmentally regulated in mammals and environmentally regulated in lower eukaryotes. Isoforms for one of the subunits, V, in the yeast Saccharomyces cerevisiae and one of the subunits, VII, in the slime mold Dictyostelium discoideum are regulated differentially by oxygen concentration. Extensive studies with the yeast subunit V isoforms have revealed that the genes for these proteins are switched on or off at very low oxygen concentrations (0.5-1 micromol l-1 O2) and that they affect the catalytic properties of holocytochrome c oxidase differentially. By altering an internal step in electron transfer between heme a and the binuclear reaction center (composed of heme a3 and CuB), the 'hypoxic' isoform, Vb, enhances the catalytic constant three- to fourfold relative to the 'aerobic' isoform, Va. Modeling studies suggest that this occurs by an interaction between transmembrane helix VII of subunit I and the transmembrane helix in subunit V. The inverse regulation of these two isoforms allows cells to assemble different types of holoenzyme isoenzymes in response to oxygen concentration. Oxygen also regulates the level of transcription of the genes for the other nuclear-coded subunits of yeast cytochrome c oxidase and affects the level of two of the mitochondrially encoded subunits (I and II) post-transcriptionally. Thus, the level of cytochrome c oxidase activity that is produced at different oxygen tensions in yeast is determined in part by the number of holoenzyme molecules that are assembled and in part by the oxygen-regulated isoforms of subunit V. The possibility that this type of control exists in other organisms is considered.

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