Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans.

ABSTRACT: Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. This redox-driven proton pump catalyzes the four-electron reduction of molecular oxygen to water, one of the most fundamental processes in biology. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding both the mechanism of oxygen reduction and its coupling to proton pumping. Using CcO from Paracoccus denitrificans, we demonstrate that the artificial F state, classically generated by reaction with an excess of hydrogen peroxide, can be converted into a new P state (in contradiction to the conventional direction of the catalytic cycle) by addition of ammonia at pH 9. We suggest that ammonia coordinates directly to Cu(B) in the binuclear active center in this P state and discuss the chemical structures of both oxoferryl intermediates F and P. Our results are compatible with a superoxide bound to Cu(B) in the F state.

SUBMITTER: von der Hocht I

PROVIDER: S-EPMC3053992 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Dynamic water networks in cytochrome C oxidase from Paracoccus denitrificans investigated by molecular dynamics simulations.

Project description:We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water molecules and their corresponding hydrogen-bonded networks inside cytochrome c oxidase. The majority of the water molecules had residence times shorter than 100 ps, but a few water molecules are fixed inside the protein for up to 1.125 ns. The hydrogen-bonded network in cytochrome c oxidase is not uniformly distributed, and the degree of water arrangement is variable. The average number of solvent sites in the proton-conducting K- and D-pathways was determined. In contrast to single water files in narrow geometries we observe significant diffusion of individual water molecules along these pathways. The highly fluctuating hydrogen-bonded networks, combined with the significant diffusion of individual water molecules, provide a basis for the transfer of protons in cytochrome c oxidase, therefore leading to a better understanding of the mechanism of proton pumping.

| S-EPMC1304046 | biostudies-literature

Modulation of the active site conformation by site-directed mutagenesis in cytochrome c oxidase from Paracoccus denitrificans.

Project description:The structural and functional properties of active site mutants of cytochrome c oxidase from Paracoccus denitrificans (PdCcO) were investigated with resonance Raman spectroscopy. Based on the Fe-CO stretching modes and low frequency heme modes, two conformers (alpha- and beta-forms) were identified that are in equilibrium in the enzyme. The alpha-conformer, which is the dominant species in the wild-type enzyme, has a shorter heme a(3) iron-Cu(B) distance and a more distorted heme, as compared to the beta-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the alpha-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the alpha-conformer is the active form of the enzyme. The data suggest that the native structure of the enzyme is in a delicate balance of intramolecular interactions. We present a model in which the mutations destabilize the alpha-conformer, with respect to the beta-conformer, and raise the activation barrier for the inter-conversion between the two conformers. The accessibility of the two conformers in the conformational space of CcO plausibly plays a critical role in coupling the redox reaction to proton translocation during the catalytic cycle of the enzyme.

| S-EPMC3418673 | biostudies-literature

Structure at 2.7 A resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment.

Project description:The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-beta-D-maltoside and cyclohexyl-hexyl-beta-D-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 A (1 A = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 A using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

| S-EPMC23397 | biostudies-literature

Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics.

Project description:Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k(12) became very rapid (3 x 10(9) M(-1) s(-1)) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ru(z)-23-c(552F) and c(1CF) was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 microM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru(z)-39-Cc formed a tight 1:1 complex with cyt c(1CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s(-1). A group of cyt c(1CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.

| S-EPMC2645956 | biostudies-literature

Titration behavior of residues at the entrance of the D-pathway of cytochrome c oxidase from paracoccus denitrificans investigated by continuum electrostatic calculations.

Project description:Continuum electrostatic calculations were employed to investigate the titration curves of the fully oxidized state of wild type and several variants of cytochrome c oxidase from Paracoccus denitrificans (N131D, N131C, N131V, and D124N) for different values of the dielectric constant of the protein. The effects of the mutations at the entrance of the D-proton transfer pathway were found to be quite localized to their immediate surroundings. The results can be well interpreted in the light of the available biochemical and structural data and help understanding the effects of mutations on proton conductivity. The mutations of aspartic acid Asp-I-124 to a neutral residue resulted in a decreased pK(a) value of His-I-28 suggesting that the mutation of His-I-28 may have a significant influence on the coupling of electron and proton transfer in cytochrome c oxidase. We also investigated the effect of the mutations N131D, N131C, and N131V on the residue Glu-I-278 in terms of its pK(a) value and electrostatic interaction energies.

| S-EPMC1366733 | biostudies-literature

The coupling of electron transfer and proton translocation: electrostatic calculations on Paracoccus denitrificans cytochrome c oxidase.

Project description:We have calculated the electrostatic potential and interaction energies of ionizable groups and analyzed the response of the protein environment to redox changes in Paracoccus denitrificans cytochrome c oxidase by using a continuum dielectric model and finite difference technique. Subsequent Monte Carlo sampling of protonation states enabled us to calculate the titration curves of all protonatable groups in the enzyme complex. Inclusion of a model membrane allowed us to restrict the calculations to the functionally essential subunits I and II. Some residues were calculated to have complex titration curves, as a result of strong electrostatic coupling, desolvation, and dipolar interactions. Around the heme a3-CuB binuclear center, we have identified a cluster of 18 strongly interacting residues that account for most of the proton uptake linked to electron transfer. This was calculated to be between 0.7 and 1.1 H+ per electron, depending on the redox transition considered. A hydroxide ion bound to CuB was determined to become protonated to form water upon transfer of the first electron to the binuclear site. The bulk of the protonation changes linked to further reduction of the heme a3-CuB center was calculated to be due to proton uptake by the interacting cluster and Glu(II-78). Upon formation of the three-electron reduced state (P1), His325, modeled in an alternative orientation away from CuB, was determined to become protonated. The agreement of these results with experiment and their relevance in the light of possible mechanisms of redox-coupled proton transfer are discussed.

| S-EPMC1302552 | biostudies-other

Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans.

Project description:The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.

| S-EPMC1134525 | biostudies-other

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222.

Project description:Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.

| S-EPMC5987163 | biostudies-literature

Transcriptome analysis of Paracoccus denitrificans during Cu-limitation

Project description:Transcriptional profiling of Paracoccus denitrificans PD1222 wild type grown to mid-exponential phase in minimal media with either 13 uM (Cu-H) or 0.5 uM (Cu-L) Cu regimes. The goal was to define the effects of Cu-limitation on denitrification genes

2013-11-18 | GSE48577 | GEO

Characterization of a TIR-like protein from Paracoccus denitrificans.

Project description:Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a alpha-helical coiled-coil. The NMR data indicates that the C-terminal TIR-like domain folds into a compact protein. Finally, using GST pull-down experiments, we show that the bacteria TIR-like domain binds to the mammalian receptor (TLR4) and adaptor (MyD88) TIR domains. We postulate that prokaryotic pathogens utilize the TIR-like proteins to interfere with the innate immune response of the mammalian host so that the bacterial infection can progress undetected.

| S-EPMC1880877 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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