Browse
Submit Data
Databases
API
Help

Dataset Information

12 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

NMR structural analysis of the yeast cytochrome c oxidase subunit Cox13 and its interaction with ATP.

ABSTRACT:

Background

Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker's yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex.

Results

Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility.

Conclusions

Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

SUBMITTER: Zhou S

PROVIDER: S-EPMC8111780 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase.

Project description:Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (?cox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient. Previous studies have reported that respiratory growth could be restored by combining ?cox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants, 5B isoenzyme expression level was 30-50% of wild-type (5A isoenzyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isoenzyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild-type levels of the 5B isoenzyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isoenzymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence the elevated activity of the 5B isoenzyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved.

| S-EPMC4255728 | biostudies-literature

Introduction of the chloroplast redox regulatory region in the yeast ATP synthase impairs cytochrome c oxidase.

Project description:The ATP synthase is under a number of mechanisms of regulation. The chloroplast ATPase has a unique mode of regulation in which activity is controlled by the redox state in the organelle. This mode of regulation is determined by a small unique region within the gamma-subunit and this region contains two cysteine residues. Introduction of this region within the yeast gamma-subunit causes a defect in oxidative phosphorylation. Oxidative phosphorylation is restored if the cysteine residues are replaced with serine. Biochemical analysis of the chimeric mitochondrial ATPase indicates that the ATP synthase is not largely altered with the cysteine residues in either the oxidized or reduced states. However, the level and activity of cytochrome c oxidase are decreased by about 90%, whereas that of NADH dehydrogenase and cytochrome c reductase are unchanged as compared with the wild-type enzymes. The level and activity of cytochrome c oxidase are restored with replacement of the cysteine residues with serine in the regulatory region. These results indicate that the chimeric ATP synthase containing cysteine, but not serine, decreases the expression or assembly of cytochrome c oxidase with little effect on the activity of the ATP synthase.

| S-EPMC2583308 | biostudies-literature

Polymorphic variation in cytochrome oxidase subunit genes.

Project description:Cytochrome oxidase (COX) activity varies between individuals and low activities associate with Alzheimer's disease. Whether genetic heterogeneity influences function of this multimeric enzyme is unknown. To explore this we sequenced three mitochondrial DNA (mtDNA) and ten nuclear COX subunit genes from at least 50 individuals. 20% had non-synonymous mtDNA COX gene polymorphisms, 12% had a COX4I1 non-synonymous G to A transition, and other genes rarely contained non-synonymous polymorphisms. Frequent untranslated region (UTR) polymorphisms were seen in COX6A1, COX6B1, COX6C, and COX7A1; heterogeneity in a COX7A1 5' UTR Sp1 site was extensive. Synonymous polymorphisms were common and less frequent in the more conserved COX1 than the less conserved COX3, suggesting at least in mtDNA synonymous polymorphisms experience selection pressure and are not functionally silent. Compound gene variations occurred within individuals. To test whether variations could have functional consequences, we studied the COX4I1 G to A transition and an AGCCCC deletion in the COX7A1 5' UTR Sp1 site. Cells expressing the COX4I1 polymorphism had reduced COX Vmax activity. In reporter construct-transduced cells where green fluorescent protein expression depended on the COX7A1 Sp1 site, AGCCCC deletion reduced fluorescence. Our findings indicate COX subunit gene heterogeneity is pervasive and may mediate COX functional variation.

| S-EPMC2910218 | biostudies-literature

Synthesis of cytochrome c oxidase subunit 1 is translationally downregulated in the absence of functional F1F0-ATP synthase.

Project description:The mitochondrial F(1)F(0)-ATP synthase or ATPase is a key enzyme for aerobic energy production in eukaryotic cells. Mutations in ATPase structural and assembly genes are the primary cause of severe human encephalomyopathies, frequently associated with a pleiotropic decrease in cytochrome c oxidase (COX) activity. We have studied the structural and functional constraints underlying the COX defect using Saccharomyces cerevisiae genetic and pharmacological models of ATPase deficiency. In both yeast Deltaatp10 and oligomycin-treated wild type cells, COX assembly is selectively impaired in the absence of functional ATPase. The COX biogenesis defect does not involve a primary alteration in the expression of the COX subunits as previously suggested but in their maturation and/or assembly. Expression of COX subunit 1, however, is translationally regulated as in most bona fide COX assembly mutants. Additionally, the COX defect in oligomycin-inhibited ATPase-deficient yeast cells, but not in atp10 cells could be partially prevented by partially dissipating the mitochondrial membrane potential using the uncoupler CCCP. Similar results were obtained with oligomycin-treated and ATP12-deficient human fibroblasts respectively. Our findings imply that fully assembled ATPase and its proton pumping function are both required for COX biogenesis in yeast and mammalian cells through a mechanism independent of Cox1p synthesis.

| S-EPMC2764804 | biostudies-literature

The Cox3p assembly module of yeast cytochrome oxidase.

Project description:Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome.

| S-EPMC3967980 | biostudies-literature

Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1.

Project description:Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3?Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A-involved in allosteric inhibition by ATP-may arise from its association with CIII. The CIII-CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.

| S-EPMC6330080 | biostudies-literature

Amyloid-? peptide binds to cytochrome C oxidase subunit 1.

Project description:Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that A? 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to A? 1-42 in ELISA as well as to A? aggregates present in AD brain. A? 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the A? 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between A? 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

| S-EPMC3424232 | biostudies-literature

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase.

Project description:The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by (1)H-(15)N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field (1)H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by (1)H- and (1)H, (15)N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.

| S-EPMC3145682 | biostudies-literature

Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast.

Project description:Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is still poorly understood. Its study in yeast has been so far impeded by the rapid turnover of unassembled subunits of the enzyme. In the present study, immunoblot analysis of blue native gels of yeast wild-type and Cox2p mutants revealed five cytochrome oxidase complexes or subcomplexes: a, b, c, d and f; a is likely to be the fully assembled enzyme; b lacks Cox6ap; d contains Cox7p and/or Cox7ap; f represents unassembled Cox1p; and c, observed only in the Cox2p mutants, contains Cox1p, Cox3p, Cox5p and Cox6p and lacks the other subunits. The identification of these novel cytochrome oxidase subcomplexes should encourage the reexamination of other yeast mutants.

| S-EPMC1199664 | biostudies-other

Structure of the intact 14-subunit human cytochrome c oxidase.

Project description:Respiration is one of the most basic features of living organisms, and the electron transport chain complexes are probably the most complicated protein system in mitochondria. Complex-IV is the terminal enzyme of the electron transport chain, existing either as randomly scattered complexes or as a component of supercomplexes. NDUFA4 was previously assumed as a subunit of complex-I, but recent biochemical data suggested it may be a subunit of complex-IV. However, no structural evidence supporting this notion was available till now. Here we obtained the 3.3?Å resolution structure of complex-IV derived from the human supercomplex I1III2IV1 and assigned the NDUFA4 subunit into complex-IV. Intriguingly, NDUFA4 lies exactly at the dimeric interface observed in previously reported crystal structures of complex-IV homodimer which would preclude complex-IV dimerization. Combining previous structural and biochemical data shown by us and other groups, we propose that the intact complex-IV is a monomer containing 14 subunits.

| S-EPMC6170408 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data