68 results on '"CYTOCHROMES"'
Search Results
2. HUMAN HEART CYTOCHROME C. CHYMOTRYPTIC PEPTIDES, TRYPTIC PEPTIDES, AND THE COMPLETE AMINO ACID SEQUENCE.
- Author
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MATSUBARA H and SMITH EL
- Subjects
- Amino Acid Sequence, Amino Acids, Chemical Phenomena, Chemistry, Chymotrypsin, Cytochromes, Cytochromes c, Heart, Myocardium, Peptides, Research, Trypsin
- Published
- 1963
3. MACROION INTERACTIONS INVOLVING CYTOCHROME SYSTEM COMPONENTS. II. CHARGE DENSITY EFFECTS ON MACROCATION INHIBITION OF CYTOCHROME OXIDASE ACTIVITY AND ON BLOCK AND REVERSAL OF SUCH INHIBITION BY MACROANIONS.
- Author
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PERSON P, MORA PT, and FINE AS
- Subjects
- Chemical Phenomena, Chemistry, Cytochromes, Electron Transport Complex IV, Enzyme Inhibitors, Ions, Polymers, Research, Spectrophotometry
- Published
- 1963
4. KINETIC OBSERVATIONS ON THE NEAR INFRARED BAND OF CYTOCHROME C OXIDASE.
- Author
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GIBSON QH and GREENWOOD C
- Subjects
- Chemical Phenomena, Chelating Agents, Chemistry, Cytochromes, Electron Transport Complex IV, Infrared Rays, Kinetics, Research, Spectrophotometry
- Published
- 1965
5. CYTOCHROME C OXIDASE COMPONENTS.III. SPECTRAL PROPERTIES OF CYTOCHROMES A AND A3.
- Author
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HORIE S and MORRISON M
- Subjects
- Chemical Phenomena, Chemistry, Cytochromes, Cytochromes a, Electron Transport Complex IV, Research
- Published
- 1963
6. CHARACTERIZATION OF A NITRATE REDUCTASE FROM THE CHEMOAUTOTROPH NITROBACTER AGILIS.
- Author
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STRAAT PA and NASON A
- Subjects
- Chemical Phenomena, Chemistry, Chemistry Techniques, Analytical, Colorimetry, Cytochromes, Nitrate Reductase, Nitrate Reductases, Nitrates, Nitrites, Nitrobacter, Oxidoreductases, Research, Spectrophotometry, Ultracentrifugation
- Published
- 1965
7. CYTOCHROME C OXIDASE COMPONENTS. IV. CHEMICAL DISTINCTION BETWEEN CYTOCHROMES ALPHA- AND ALPHA-3.
- Author
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MORRISON M and HORIE S
- Subjects
- Chemical Phenomena, Borates, Chemistry, Chromatography, Cyanides, Cytochromes, Electron Transport Complex IV, Heme, Research, Spectrophotometry, Sulfites, Urea
- Published
- 1964
8. CYTOCHROME C OXIDASE COMPONENTS. VII. AN EVALUATION OF THE CARBON MONOXIDE-COMBINING CAPACITY IN ORDER TO DETERMINE THE STOICHIOMETRY OF CYTOCHROMES A AND A3.
- Author
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MORRISON M and HORIE S
- Subjects
- Carbon Monoxide, Chemical Phenomena, Chemistry, Cytochromes, Cytochromes a, Electron Transport Complex IV, Heme, Hemoglobins, Research, Spectrum Analysis
- Published
- 1965
9. DIHYDROOROTIC DEHYDROGENASE. II. OXIDATION AND REDUCTION OF CYTOCHROME C.
- Author
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MILLER RW and MASSEY V
- Subjects
- Bacteria, Chelating Agents, Chemical Phenomena, Chemistry, Cytochromes, Cytochromes c, Hydrogen Peroxide, NAD, Oxidation-Reduction, Oxidoreductases, Research, Spectrum Analysis, Sulfonic Acids
- Published
- 1965
10. THE REACTION OF CYTOCHROME OXIDASE WITH CYTOCHROME C.
- Author
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GIBSON QH and GREENWOOD C
- Subjects
- Kinetics, Ascorbic Acid, Carbon Monoxide, Chemical Phenomena, Chemistry, Cytochromes, Cytochromes c, Electron Transport, Electron Transport Complex IV, Hydroquinones, Phenols, Research, Spectrophotometry
- Published
- 1965
11. MACROION INTERACTIONS INVOLVING COMPONENTS OF THE CYTOCHROME SYSTEM. IV. POLYION-INDUCED REVERSIBLE OXIDATIONS AND REDUCTIONS OF CYTOCHROME C.
- Author
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PERSON P, FINE AS, MORA PT, and ZIPPER H
- Subjects
- Aspartic Acid, Chemical Phenomena, Chemistry, Cytochromes, Cytochromes c, Glucose, Glutamates, Heparin, Ions, Lysine, Oxidation-Reduction, Peptides, Polysaccharides, Research, Sulfates
- Published
- 1965
12. Yeast Cells Lacking the Mitochondrial Gene Encoding the ATP Synthase Subunit 6 Exhibit a Selective Loss of Complex IV and Unusual Mitochondrial Morphology.
- Author
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Rak, Malgorzata, Tetaud, Emmanuel, Godard, François, Sagot, Isabelle, Salin, Bénédicte, Duvezin-Caubet, Stéphane, Slonimski, Piotr P., Rytka, Joanna, and di Rago, Jean-Paul
- Subjects
- *
ADENOSINE triphosphatase , *MITOCHONDRIAL DNA , *CYTOCHROMES , *MITOCHONDRIA , *BIOMARKERS , *CHEMISTRY , *BIOLOGY , *BIOCHEMISTRY - Abstract
Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of ρ-/ρ0 petites issued from large deletions in mtDNA could be restricted to 20–30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
13. The Efficient Functioning of Photosynthesis and Respiration in Synechocystis sp. PCC 6803 Strictly Requires the Presence of either cytochrome c6 or Plastocyanin.
- Author
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Durán, Raúl V., Hervás, Manuel, De la Rosa, Miguel A., and Navarro, José A.
- Subjects
- *
CYANOBACTERIA , *CYTOCHROMES , *BIOCHEMISTRY , *BIOLOGY , *CHEMISTRY , *MEDICAL sciences - Abstract
In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the pete or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wildtype cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
14. Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome
- Author
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Christina Ferousi, Joachim Reimann, Frauke Baymann, Simon Lindhoud, Eric R. Hester, Boran Kartal, Radboud university [Nijmegen], Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute for Marine Microbiology, Max-Planck-Gesellschaft, and Radboud University [Nijmegen]
- Subjects
0301 basic medicine ,Hemeprotein ,Heme binding ,Cytochrome ,Stereochemistry ,[SDV]Life Sciences [q-bio] ,Amino Acid Motifs ,Heme ,contracted heme binding motif ,Biochemistry ,tetraheme cytochrome ,03 medical and health sciences ,chemistry.chemical_compound ,hydrazine synthase (HZS) ,Gene cluster ,nitrogen cycle ,[CHIM]Chemical Sciences ,Life Science ,Amino Acid Sequence ,nitric oxide (NO) ,Molecular Biology ,Bacteria ,030102 biochemistry & molecular biology ,biology ,ATP synthase ,Chemistry ,Cytochrome c ,Cell Biology ,heme proteins ,KsTH ,cytochrome c ,030104 developmental biology ,Anammox ,Ecological Microbiology ,Enzymology ,biology.protein ,Cytochromes ,anaerobic ammonium oxidation (anammox) ,Oxidation-Reduction ,Protein Binding ,EPR spectroscopy - Abstract
International audience; Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally occurring contracted Cys–Lys–Cys–His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named here KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.
- Published
- 2019
- Full Text
- View/download PDF
15. A unique aromatic residue modulates the redox range of a periplasmic multiheme cytochrome from Geobacter metallireducens
- Author
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Pilar C. Portela, Marta A. Silva, Liliana R. Teixeira, and Carlos A. Salgueiro
- Subjects
Models, Molecular ,CIL, Cambridge Isotope Laboratories ,0301 basic medicine ,Cytochrome ,Stereochemistry ,Heme ,bioenergetics ,Biochemistry ,Redox ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Redox titration ,Aromatic amino acids ,Molecular Biology ,Geobacter sulfurreducens ,030102 biochemistry & molecular biology ,biology ,HSQC, heteronuclear single quantum coherence ,Cell Biology ,Periplasmic space ,Geobacter metallireducens ,electron transfer ,biology.organism_classification ,NMR ,Kinetics ,cytochrome c ,030104 developmental biology ,EXSY, EXchange SpectroscopY ,chemistry ,Periplasm ,biology.protein ,Cytochromes ,site-directed mutagenesis ,Geobacter ,Hydrophobic and Hydrophilic Interactions ,Oxidation-Reduction ,Research Article - Abstract
Geobacter bacteria are able to transfer electrons to the exterior of the cell and reduce extracellular electron acceptors including toxic/radioactive metals and electrode surfaces, with potential applications in bioremediation or electricity harvesting. The triheme c-type cytochrome PpcA from Geobacter metallireducens plays a crucial role in bridging the electron transfer from the inner to the outer membrane, ensuring an effective extracellular electron transfer. This cytochrome shares 80% identity with PpcA from Geobacter sulfurreducens, but their redox properties are markedly different, thus determining the distinctive working redox potential ranges in the two bacteria. PpcA from G. metallireducens possesses two extra aromatic amino acids (Phe-6 and Trp-45) in its hydrophobic heme core, whereas PpcA from G. sulfurreducens has a leucine and a methionine in the equivalent positions. Given the different nature of these residues in the two cytochromes, we have hypothesized that the extra aromatic amino acids could be partially responsible for the observed functional differences. In this work, we have replaced Phe-6 and Trp-45 residues by their nonaromatic counterparts in PpcA from G. sulfurreducens. Using redox titrations followed by UV–visible and NMR spectroscopy we observed that residue Trp-45 shifted the redox potential range 33% toward that of PpcA from G. sulfurreducens, whereas Phe-6 produced a negligible effect. For the first time, it is shown that the inclusion of an aromatic residue at the heme core can modulate the working redox range in abundant periplasmic proteins, paving the way to engineer bacterial strains for optimal microbial bioelectrochemical applications.
- Published
- 2021
16. The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways
- Author
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Norbert Krauß, Tilman Lamparter, Fan Zhang, Inga Oberpichler, Jacqueline Kalms, David von Stetten, and Patrick Scheerer
- Subjects
Models, Molecular ,Ultraviolet Rays ,Stereochemistry ,Pyrimidine dimer ,Crystallography, X-Ray ,Biochemistry ,Evolution, Molecular ,Deoxyribodipyrimidine photo-lyase ,chemistry.chemical_compound ,Cryptochrome ,Enzyme Stability ,Binding site ,Site-directed mutagenesis ,Photolyase ,Molecular Biology ,Tetrahydrofolates ,Flavin adenine dinucleotide ,Binding Sites ,Cell Biology ,Chromophore ,Protein Structure, Tertiary ,chemistry ,Agrobacterium tumefaciens ,Pyrimidine Dimers ,Protein Structure and Folding ,Flavin-Adenine Dinucleotide ,Cytochromes ,Nucleic Acid Conformation ,Deoxyribodipyrimidine Photo-Lyase ,Oxidation-Reduction ,DNA Damage - Abstract
Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.
- Published
- 2015
17. Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes
- Author
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Bianca Eisel, Ben C. Berks, Susan M. Lea, Steven Johnson, Daniel B. Grabarczyk, and Paul E. Chappell
- Subjects
Cytochrome ,Protein Conformation ,Stereochemistry ,Metalloenzyme ,Molecular Sequence Data ,Thiosulfates ,Bacterial Metabolism ,Heme ,Biochemistry ,Thiosulfate dehydrogenase ,Mass Spectrometry ,Electron Transport ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Thiosulfate Dehydrogenase ,Amino Acid Sequence ,Cysteine ,Sulfur Oxidizing Bacteria ,Molecular Biology ,DNA Primers ,030304 developmental biology ,Tetrathionate ,Thiosulfate ,0303 health sciences ,Bacteria ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,030302 biochemistry & molecular biology ,Active site ,Thiosulfate binding ,Cell Biology ,3. Good health ,Sox System ,chemistry ,Enzymology ,biology.protein ,Cytochromes ,Spectrophotometry, Ultraviolet ,Oxidation-Reduction - Abstract
Background: The hemoprotein TsdA catalyzes the oxidation of two thiosulfate molecules to form tetrathionate. Results: The mechanism of TsdA has been probed using biochemical and structural methods. Conclusion: The TsdA reaction proceeds via a cysteine S-thiosulfonate intermediate formed on a cysteine ligand to the active site heme. Significance: TsdA provides a catalytic model for other members of the SoxA enzyme family., Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.
- Published
- 2015
18. Transcript Profiling and Inference of Escherichia coli K-12 ArcA Activity across the Range of Physiologically Relevant Oxygen Concentrations
- Author
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Robert K. Poole, Guido Sanguinetti, Alex Ter Beek, Alison I. Graham, H. M. Shahzad Asif, Matthew D. Rolfe, Eleanor W. Trotter, Joost Teixeira de Mattos, and Jeffrey Green
- Subjects
Transcription, Genetic ,Cytochrome ,Ubiquinone ,Phosphatase ,Respiratory chain ,chemistry.chemical_element ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Oxygen ,medicine ,Gene Regulation ,Anaerobiosis ,Phosphorylation ,Molecular Biology ,Escherichia coli ,Regulation of gene expression ,Escherichia coli K12 ,Escherichia coli Proteins ,Membrane Proteins ,Cell Biology ,Metabolism ,Cytochrome b Group ,Aerobiosis ,Repressor Proteins ,Electron Transport Chain Complex Proteins ,chemistry ,biology.protein ,Cytochromes ,Oxidoreductases ,Bacterial outer membrane ,Protein Kinases ,Bacterial Outer Membrane Proteins - Abstract
Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.
- Published
- 2011
19. Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1
- Author
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Jeffrey A. Gralnick, Edward V. LaBelle, Daniel B. Baron, Daniel R. Bond, and Dan Coursolle
- Subjects
Shewanella ,Standard hydrogen electrode ,Analytical chemistry ,Cytochrome c Group ,Electrons ,Electrochemistry ,Photochemistry ,Biochemistry ,Metal ,Electron transfer ,Shewanella oneidensis ,Electrodes ,Molecular Biology ,Voltammetry ,chemistry.chemical_classification ,Microscopy, Confocal ,biology ,Chemistry ,Cell Biology ,Electron acceptor ,biology.organism_classification ,Kinetics ,Metabolism and Bioenergetics ,Metals ,Biofilms ,visual_art ,Mutation ,Microscopy, Electron, Scanning ,visual_art.visual_art_medium ,Cytochromes ,Adsorption ,Oxidation-Reduction ,Bacterial Outer Membrane Proteins - Abstract
Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at approximately 0 V (versus standard hydrogen electrode), and was altered in single (DeltaomcA, DeltamtrC) and double deletion (DeltaomcA/DeltamtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (-0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (approximately 1 s(-1)). These observations indicated that anodic current in the higher (0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.
- Published
- 2009
20. Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40phox in Activation of the Neutrophil NADPH Oxidase
- Author
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Glenn E. Brown, Mary Q. Stewart, Sarah A. Bissonnette, Christina M. Glazier, Chris D. Ellson, and Michael B. Yaffe
- Subjects
Neutrophils ,Models, Biological ,Biochemistry ,Article ,Cell Line ,src Homology Domains ,chemistry.chemical_compound ,Phosphatidylinositol Phosphates ,Multienzyme Complexes ,Superoxides ,Humans ,Phosphatidylinositol ,Molecular Biology ,Oxidase test ,NADPH oxidase ,biology ,Superoxide ,Phosphatidylinositol 3-phosphate ,NADPH Oxidases ,Cell Biology ,PX domain ,Molecular biology ,rac GTP-Binding Proteins ,Cell biology ,Enzyme Activation ,Rac GTP-Binding Proteins ,Kinetics ,chemistry ,biology.protein ,Cytochromes ,Proto-oncogene tyrosine-protein kinase Src - Abstract
In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.
- Published
- 2008
21. Characterization of Mutants That Change the Hydrogen Bonding of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli
- Author
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Robert B. Gennis, Rimma I. Samoilova, Lai Lai Yap, and Sergei A. Dikanov
- Subjects
Ubiquinol ,Semiquinone ,Cytochrome ,Stereochemistry ,Glutamine ,Ubiquinol oxidase ,Arginine ,Photochemistry ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Quinone binding ,Benzoquinones ,Escherichia coli ,Molecular Biology ,Aspartic Acid ,biology ,Hydrogen bond ,Escherichia coli Proteins ,Electron Spin Resonance Spectroscopy ,Quinones ,Wild type ,Hydrogen Bonding ,Cell Biology ,Cytochrome b Group ,Kinetics ,Models, Chemical ,Catalytic cycle ,chemistry ,Mutation ,biology.protein ,Cytochromes ,Protons ,Protein Binding - Abstract
The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.
- Published
- 2007
22. Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli
- Author
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Rimma I. Samoilova, Lai Lai Yap, Sergei A. Dikanov, and Robert B. Gennis
- Subjects
Proton ,Cytochrome ,Semiquinone ,Ubiquinol oxidase ,Electrons ,Photochemistry ,Biochemistry ,Electron Transport ,Quinone binding ,Benzoquinones ,Escherichia coli ,Molecular Biology ,Hyperfine structure ,biology ,Chemistry ,Hydrogen bond ,Escherichia coli Proteins ,Hydrogen Bonding ,Cell Biology ,Cytochrome b Group ,Kinetics ,Electron Transport Chain Complex Proteins ,Models, Chemical ,Covalent bond ,biology.protein ,Anisotropy ,Cytochromes ,Protons - Abstract
The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.
- Published
- 2006
23. A Bacterial Glutathione Transporter (Escherichia coli CydDC) Exports Reductant to the Periplasm
- Author
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Robert K. Poole, Hilary C. Robinson, and Marc S. Pittman
- Subjects
Cytoplasm ,Time Factors ,Cytochrome ,Mutant ,Swarming motility ,Lactose ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Dithiothreitol ,Substrate Specificity ,chemistry.chemical_compound ,Escherichia coli ,medicine ,Homeostasis ,Disulfides ,Molecular Biology ,Dose-Response Relationship, Drug ,Escherichia coli Proteins ,Membrane Transport Proteins ,Biological Transport ,Penicillin G ,Cell Biology ,Periplasmic space ,Glutathione ,Cytochromes b ,Cytochrome b Group ,Oxygen ,Electron Transport Chain Complex Proteins ,chemistry ,Mutation ,Periplasm ,biology.protein ,Cytochromes ,bacteria ,Glutathione disulfide ,ATP-Binding Cassette Transporters ,Vanadates ,Carrier Proteins ,Oxidoreductases ,Dimerization ,Oxidation-Reduction - Abstract
Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis.
- Published
- 2005
24. Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling
- Author
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Hiro Nakamura, Yoichi Yamazaki, Tatsushi Mogi, and Hideki Kandori
- Subjects
Models, Molecular ,Light ,Cytochrome ,Protein Conformation ,Stereochemistry ,Ubiquinol oxidase ,Glutamic Acid ,Protonation ,Ligands ,Biochemistry ,Redox ,Protein Structure, Secondary ,Electron Transport Complex IV ,chemistry.chemical_compound ,Spectroscopy, Fourier Transform Infrared ,Escherichia coli ,Animals ,Histidine ,Fourier transform infrared spectroscopy ,Molecular Biology ,Heme ,Bond cleavage ,biology ,Chemistry ,Cell Biology ,Hydrogen-Ion Concentration ,Oxygen ,Models, Chemical ,Spectrophotometry ,Covalent bond ,Potassium ,biology.protein ,Cytochromes ,Tyrosine ,Cattle ,Oxidation-Reduction ,Copper ,Hydrogen - Abstract
Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cepsilon-Nepsilon covalent bond with one of the Cu(B) ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669-6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either L-[1-13C]Tyr or L-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (approximately 1620-1680 cm(-1)) and -II ( approximately 1540-1560 cm(-1)) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-Cu(B) binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.
- Published
- 2005
25. Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures
- Author
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Euan Gordon, Stuart J. Ferguson, Richard S. Zajicek, and Myles R. Cheesman
- Subjects
Anions ,Steric effects ,Nitrite Reductases ,Cytochrome ,Ultraviolet Rays ,Stereochemistry ,Cyanide ,Cytochrome c Group ,Heme ,Crystallography, X-Ray ,Ligands ,Nitric Oxide ,Photochemistry ,Biochemistry ,chemistry.chemical_compound ,Escherichia coli ,Histidine ,Potassium Cyanide ,Anion binding ,Molecular Biology ,Nitrites ,Paracoccus pantotrophus ,Binding Sites ,biology ,Ligand ,Electron Spin Resonance Spectroscopy ,Temperature ,Active site ,Cell Biology ,Hydrogen-Ion Concentration ,Oxygen ,Kinetics ,chemistry ,Spectrophotometry ,Mutation ,biology.protein ,Cytochromes ,Tyrosine ,Protein Binding - Abstract
Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.
- Published
- 2005
26. Molecular Basis of Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes Is Revealed by High Resolution Structure of a Heterodimeric Complex of the Catalytic Molybdopterin Subunit and a c-Type Cytochrome Subunit
- Author
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Ulrike Kappler and Susan Bailey
- Subjects
Models, Molecular ,Protein Folding ,Cytochrome ,Protein Conformation ,Sulfite Dehydrogenase ,Molecular Sequence Data ,Static Electricity ,Electrons ,Heme ,Arginine ,Biochemistry ,Catalysis ,Protein Structure, Secondary ,Electron Transport ,Electron transfer ,chemistry.chemical_compound ,Sulfite ,Oxidoreductase ,Catalytic Domain ,Xanthobacter ,Sulfites ,Sulfite dehydrogenase ,Amino Acid Sequence ,Molecular Biology ,Cytochrome Reductases ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,biology ,Chemistry ,Cytochrome c ,Molybdopterin ,Gene Expression Regulation, Bacterial ,Cell Biology ,Electron transport chain ,Protein Structure, Tertiary ,Oxygen ,biology.protein ,Cytochromes - Abstract
Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 A) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.
- Published
- 2005
27. Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp
- Author
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Lígia M. Saraiva, Carlos Frazão, Ilídio J. Correia, Teresa Catarino, David L. Turner, António V. Xavier, Catarina M. Paquete, Ana Varela Coelho, Ricardo O. Louro, Cláudia C. Almeida, Maria Arménia Carrondo, and uBibliorum
- Subjects
DNA, Bacterial ,Deltaproteobacteria ,Models, Molecular ,Hydrogenase ,Cytochrome ,Stereochemistry ,Sequence analysis ,Molecular Sequence Data ,Cytochrome c Group ,Heme ,Crystallography, X-Ray ,Biochemistry ,Redox ,Electron Transport ,Residue (chemistry) ,Electron transfer ,chemistry.chemical_compound ,Bacterial Proteins ,Cloning, Molecular ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Base Sequence ,biology ,Chemistry ,Desulfomicrobium Sp ,Genetic Variation ,Cell Biology ,Hydrogen-Ion Concentration ,Electron transport chain ,NMR ,Kinetics ,Genes, Bacterial ,biology.protein ,Cytochromes ,Thermodynamics ,Protons ,Oxidation-Reduction - Abstract
The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 → Ser, Val-45 → Ala, and Phe-88 → Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.
- Published
- 2004
28. Two Distinct Binding Sites for High Potential Iron-Sulfur Protein and Cytochrome c on the Reaction Center-bound Cytochrome of Rubrivivax gelatinosus
- Author
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Jean Alric, Pierre Parot, Rainer Hienerwadel, Jean-Luc Pellequer, Makoto Yoshida, Kenji V. P. Nagashima, Shu-wen W. Chen, André Verméglio, Luminy Génétique et Biophysique des Plantes (LGBP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Tokyo Metropolitan University [Tokyo] (TMU), Service de Biochimie et Toxicologie Nucléaire (SBTN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Laboratoire des Intéractions et Reconnaissances Moléculaires (LIRM)
- Subjects
Iron-Sulfur Proteins ,Models, Molecular ,Time Factors ,Cytochrome ,Stereochemistry ,[SDV]Life Sciences [q-bio] ,Molecular Sequence Data ,Photosynthetic Reaction Center Complex Proteins ,Cytochrome c Group ,Electrons ,Biochemistry ,Protein Structure, Secondary ,Bacterial Proteins ,Species Specificity ,Cytochrome C1 ,Cytochrome c oxidase ,Amino Acid Sequence ,Photosynthesis ,Ferricyanides ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,Binding Sites ,Base Sequence ,biology ,Burkholderiaceae ,Chemistry ,Cytochrome c peroxidase ,Cytochrome b6f complex ,Cytochrome c ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Cytochromes c ,Cytochrome P450 reductase ,Cell Biology ,Protein Structure, Tertiary ,Oxygen ,Kinetics ,Spectrophotometry ,Coenzyme Q – cytochrome c reductase ,Mutation ,biology.protein ,Cytochromes ,Cell Division - Abstract
The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.
- Published
- 2004
29. Structural and Functional Characterization of the Unusual Triheme Cytochrome Bound to the Reaction Center of Rhodovulum sulfidophilum
- Author
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Barbara Schoepp-Cothenet, Makoto Yoshida, Rainer Hienerwadel, Keizo Shimada, André Verméglio, Wolfgang Nitschke, Katsumi Matsuura, Jean Alric, Yusuke Tsukatani, and Kenji V. P. Nagashima
- Subjects
Photosynthetic reaction centre ,Rhodovulum ,Cytochrome ,Stereochemistry ,Amino Acid Motifs ,Heme ,Ligands ,Models, Biological ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cysteine ,Molecular Biology ,Binding Sites ,Methionine ,Models, Genetic ,biology ,Ligand ,Mutagenesis ,Electron Spin Resonance Spectroscopy ,Cell Biology ,biology.organism_classification ,chemistry ,Spectrophotometry ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Cytochromes ,Oxidation-Reduction ,Cell Division ,Plasmids ,Protein Binding - Abstract
The cytochrome bound to the photosynthetic reaction center of Rhodovulum sulfidophilum presents two unusual characteristics with respect to the well characterized tetraheme cytochromes. This cytochrome contains only three hemes because it lacks the peptide motif CXXCH, which binds the most distal fourth heme. In addition, we show that the sixth axial ligand of the third heme is a cysteine (Cys-148) instead of the usual methionine ligand. This ligand exchange results in a very low midpoint potential (-160 +/- 10 mV). The influence of the unusual cysteine ligand on the midpoint potential of this distal heme was further investigated by site-directed mutagenesis. The midpoint potential of this heme is upshifted to +310 mV when cysteine 148 is replaced by methionine, in agreement with the typical redox properties of a His/Met coordinated heme. Because of the large increase in the midpoint potential of the distal heme in the mutant, both the native and modified high potential hemes are photooxidized at a redox poise where only the former is photooxidizable in the wild type. The relative orientation of the three hemes, determined by EPR measurements, is shown different from tetraheme cytochromes. The evolutionary basis of the concomitant loss of the fourth heme and the down-conversion of the third heme is discussed in light of phylogenetic relationships of the Rhodovulum species triheme cytochromes to other reaction center-associated tetraheme cytochromes.
- Published
- 2004
30. Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase
- Author
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Pierre Moënne-Loccoz, Christina Divne, Michael H. Gold, B. Martin Hallberg, Frederik A.J. Rotsaert, V. Renganathan, and Simon de Vries
- Subjects
Models, Molecular ,Cellobiose dehydrogenase ,Hemeprotein ,Cytochrome ,Protein Conformation ,Stereochemistry ,Iron ,Blotting, Western ,Electrons ,Heme ,Crystallography, X-Ray ,Ligands ,Phanerochaete ,Spectrum Analysis, Raman ,Biochemistry ,chemistry.chemical_compound ,Electrochemistry ,Molecular Biology ,biology ,Cytochrome b ,Cytochrome c ,Wild type ,Cell Biology ,Hydrogen-Ion Concentration ,Protein Structure, Tertiary ,Oxygen ,Kinetics ,Heme B ,chemistry ,Spectrophotometry ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,Cytochromes ,Carbohydrate Dehydrogenases ,Electrophoresis, Polyacrylamide Gel ,Oxidation-Reduction ,Plasmids - Abstract
The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.
- Published
- 2003
31. A Complex II Defect Affects Mitochondrial Structure, Leading to ced-3- and ced-4-dependent Apoptosis and Aging
- Author
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Nanami Senoo-Matsuda, Shinichi Yoshimura, Philip S. Hartman, Naoaki Ishii, and Akira Akatsuka
- Subjects
Aging ,Time Factors ,Protein subunit ,Blotting, Western ,Mutant ,Apoptosis ,Biology ,medicine.disease_cause ,Models, Biological ,Biochemistry ,Membrane Potentials ,chemistry.chemical_compound ,Oxidoreductase ,Proto-Oncogene Proteins ,medicine ,Animals ,Myocyte ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Hypoxia ,Molecular Biology ,Hyperoxia ,chemistry.chemical_classification ,Superoxide ,Calcium-Binding Proteins ,Antibodies, Monoclonal ,Cell Biology ,Cytochromes b ,biology.organism_classification ,Immunohistochemistry ,Mitochondria ,Cell biology ,Oxygen ,Microscopy, Electron ,Microscopy, Fluorescence ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,Caspases ,Mutation ,Cytochromes ,medicine.symptom ,Apoptosis Regulatory Proteins ,Oxidative stress - Abstract
The mev-1(kn1) mutation of Caenorhabditis elegans is in Cyt-1, which encodes a subunit of succinate-coenzyme Q oxidoreductase in the mitochondrial electron transport chain. Mutants are hypersensitive to oxidative stress and age precociously in part because of increased superoxide anion production. Here, we show that mev-1 mutants are defective in succinate-coenzyme Q oxidoreductase, possess ultrastructural mitochondrial abnormalities (especially in muscle cells), show a loss of membrane potential, have altered CED-9 and Cyt-1 protein levels under hyperoxia, and contain ced-3-and ced-4-dependent supernumerary apoptotic cells. These defects likely explain the failure of mev-1 to complete embryonic development under hyperoxia as well as its reduced life span.
- Published
- 2003
32. Structure and Kinetic Properties of Paracoccus pantotrophus Cytochrome cd1 Nitrite Reductase with the d1 Heme Active Site Ligand Tyrosine 25 Replaced by Serine
- Author
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Janos Hajdu, Christopher W. Higham, Carsten D. Richter, Euan H. J. Gordon, Tove Sjögren, Vilmos Fülöp, James W. A. Allen, Stuart J. Ferguson, and Malin Löfqvist
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Nitrite Reductases ,Stereochemistry ,Heme ,Reductase ,Ligands ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Cofactor ,Electron Transport Complex IV ,chemistry.chemical_compound ,Hydroxylamine ,Serine ,Molecular Biology ,Histidine ,Paracoccus pantotrophus ,Binding Sites ,biology ,Active site ,Cytochrome P450 reductase ,Gene Expression Regulation, Bacterial ,Paracoccus ,Cell Biology ,Protein Structure, Tertiary ,Kinetics ,chemistry ,Mutagenesis ,biology.protein ,Cytochromes ,Tyrosine - Abstract
The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.
- Published
- 2003
33. Nitric Oxide Reacts with the Single-electron Reduced Active Site of Cytochrome c Oxidase
- Author
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Bernd Ludwig, Alessandro Giuffrè, Francesco Malatesta, Maurizio Brunori, Emilio D'Itri, Maria Cecilia Barone, Paolo Sarti, and Hans-Werner Müller
- Subjects
Time Factors ,Negibacteria ,Photochemistry ,Biochemistry ,chemistry.chemical_compound ,Models ,enzyme kinetics ,cytochrome c oxidase ,enzyme inhibition ,Bacteria (microorganisms) ,Heme ,chemistry.chemical_classification ,biology ,article ,Hydrogen-Ion Concentration ,enzyme activity ,priority journal ,Spectrophotometry ,enzyme active site ,reaction kinetics ,Nitrogen oxides ,Protein Binding ,Stereochemistry ,reduction ,Chemical ,Electrons ,Nitric Oxide ,Electron Transport Complex IV ,Reaction rate constant ,methenamine ,Cytochrome c oxidase ,enzyme mechanism ,mutant ,Binding site ,ruthenium ,Molecular Biology ,Paracoccus denitrificans ,nonhuman ,Binding Sites ,Active site ,Cell Biology ,biology.organism_classification ,enzyme metabolism ,Kinetics ,Enzyme ,Heme A ,Models, Chemical ,chemistry ,biology.protein ,Cytochromes - Abstract
The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.
- Published
- 2002
34. Heme Ligation and Conformational Plasticity in the Isolatedc Domain of Cytochrome cd 1 Nitrite Reductase
- Author
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E. Steensma, Janos Hajdu, Euan H. J. Gordon, Linda M. Öster, and Stuart J. Ferguson
- Subjects
Hemeproteins ,Models, Molecular ,Protein Denaturation ,Hot Temperature ,Nitrite Reductases ,Stereochemistry ,Heme ,Ligands ,Photochemistry ,Biochemistry ,Redox ,Electron Transport Complex IV ,chemistry.chemical_compound ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Cytochrome c peroxidase ,Ligand ,Cytochrome P450 reductase ,Paracoccus ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Nitrite reductase ,Peptide Fragments ,Protein tertiary structure ,Protein Structure, Tertiary ,Solutions ,chemistry ,Spectrophotometry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Cytochromes ,Protons ,Oxidation-Reduction - Abstract
The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.
- Published
- 2001
35. Flavohemoglobin Hmp Affords Inducible Protection for Escherichia coli Respiration, Catalyzed by Cytochromesbo′ or bd, from Nitric Oxide
- Author
-
Catherine E. Mills, Nikolaos Ioannidis, Tania M. Stevanin, Robert K. Poole, Martin N. Hughes, and Sung Oog Kim
- Subjects
Hemeproteins ,Nitroprusside ,animal structures ,Cellular respiration ,chemistry.chemical_element ,Microbial Sensitivity Tests ,Biology ,Nitric Oxide ,Biochemistry ,Oxygen ,Nitric oxide ,chemistry.chemical_compound ,Oxygen Consumption ,Bacterial Proteins ,Dihydropteridine Reductase ,Respiration ,Escherichia coli ,medicine ,Extracellular ,NADH, NADPH Oxidoreductases ,Molecular Biology ,Oxidase test ,Cyanides ,Dose-Response Relationship, Drug ,Escherichia coli Proteins ,Nitric oxide dioxygenase ,Cell Biology ,Cytochrome b Group ,Glutathione ,Electron Transport Chain Complex Proteins ,chemistry ,Spectrophotometry ,Mutation ,S-Nitrosoglutathione ,Cytochromes ,Sodium nitroprusside ,Oxidoreductases ,Cell Division ,Nitroso Compounds ,medicine.drug - Abstract
Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments.
- Published
- 2000
36. Time-resolved Infrared Spectroscopy Reveals a Stable Ferric Heme-NO Intermediate in the Reaction of Paracoccus pantotrophus Cytochrome cd 1 Nitrite Reductase with Nitrite
- Author
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Roger N. F. Thorneley, Simon J. George, Stuart J. Ferguson, and James W. A. Allen
- Subjects
Nitrite Reductases ,Time Factors ,Cytochrome ,Stereochemistry ,Iron ,Cytochrome c Group ,Heme ,Nitric Oxide ,Photochemistry ,Biochemistry ,chemistry.chemical_compound ,Spectroscopy, Fourier Transform Infrared ,medicine ,Nitrite ,Molecular Biology ,Nitrites ,Paracoccus pantotrophus ,biology ,Chemistry ,Active site ,Paracoccus ,Cell Biology ,Nitrite reductase ,Respiratory enzyme ,biology.protein ,Cytochromes ,Ferric ,Oxidation-Reduction ,medicine.drug - Abstract
Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant. The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1). This d(1) heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+). No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd(1) are discussed.
- Published
- 2000
37. X-ray Crystallographic Study of Cyanide Binding Provides Insights into the Structure-Function Relationship for Cytochromecd 1 Nitrite Reductase from Paracoccus pantotrophus
- Author
-
Stuart J. Ferguson, Arif Jafferji, Vilmos Fülöp, and James W. A. Allen
- Subjects
Anions ,Models, Molecular ,Nitrite Reductases ,Protein Conformation ,Cyanide ,Dimer ,Heme ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Electron Transport Complex IV ,Structure-Activity Relationship ,chemistry.chemical_compound ,medicine ,Molecular Biology ,Paracoccus pantotrophus ,Binding Sites ,Cyanides ,biology ,Ligand ,Hydrogen bond ,Active site ,Hydrogen Bonding ,Paracoccus ,Cell Biology ,Kinetics ,Crystallography ,chemistry ,biology.protein ,Cytochromes ,Ferric ,Oxidation-Reduction ,Protein Binding ,medicine.drug - Abstract
We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.
- Published
- 2000
38. Induction of Coproporphyrinogen Oxidase inChlamydomonas Chloroplasts Occurs via Transcriptional Regulation of Cpx1 Mediated by Copper Response Elements and Increased Translation from a Copper Deficiency-specific Form of the Transcript
- Author
-
Sabeeha S. Merchant, Jeanette M. Quinn, and Stacie S. Nakamoto
- Subjects
Chloroplasts ,Nuclear gene ,Transcription, Genetic ,Molecular Sequence Data ,Response element ,Chlamydomonas reinhardtii ,Biology ,Biochemistry ,Gene Expression Regulation, Enzymologic ,chemistry.chemical_compound ,Coproporphyrinogen Oxidase ,Gabaculine ,Genes, Reporter ,Transcriptional regulation ,Animals ,Amino Acid Sequence ,Metalloexopeptidases ,Molecular Biology ,Gene ,Arylsulfatases ,Coproporphyrinogen III oxidase ,Base Sequence ,Metalloendopeptidases ,DNA ,Cell Biology ,biology.organism_classification ,Cytochromes f ,Blotting, Southern ,chemistry ,Protein Biosynthesis ,Cytochromes ,Carrier Proteins ,Copper - Abstract
Coproporphyrinogen III oxidase, encoded by a single nuclear gene in Chlamydomonas reinhardtii, produces three distinct transcripts. One of these transcripts is greatly induced in copper-deficient cells by transcriptional activation, whereas the other forms are copper-insensitive. The induced form of the transcript was expressed coordinately with the cytochrome c6-encoding (Cyc6) gene, which is known to be transcriptionally regulated in copper-deficient cells. The sequence GTAC, which forms the core of a copper response element associated with the Cyc6 gene, is also essential for induction of the Cpx1 gene, suggesting that both are targets of the same signal transduction pathway. The constitutive and induced Cpx1 transcripts have the same half-lives in vivo, and all encode the same polypeptide with a chloroplast-targeting transit sequence, but the shortest one representing the induced form is a 2-4-fold better template for translation than are either of the constitutive forms. The enzyme remains localized to a soluble compartment in the chloroplast even in induced cells, and its abundance is not affected when the tetrapyrrole pathway is manipulated either genetically or by gabaculine treatment.
- Published
- 1999
39. Hydrophobic Core but Not Amino-terminal Charged Residues Are Required for Translocation of an Integral Thylakoid Membrane Protein in Vivo
- Author
-
Bruce D. Kohorn and Benoit Baillet
- Subjects
Signal peptide ,Chloroplasts ,Molecular Sequence Data ,Biological Transport, Active ,Protein Sorting Signals ,Biochemistry ,Residue (chemistry) ,Suppression, Genetic ,Electrochemistry ,Animals ,Amino Acid Sequence ,Molecular Biology ,Integral membrane protein ,chemistry.chemical_classification ,Cytochrome f ,Molecular Structure ,biology ,Chlamydomonas ,Membrane Proteins ,Cell Biology ,biology.organism_classification ,Cytochromes f ,Amino acid ,chemistry ,Thylakoid ,Mutation ,Cytochromes ,Leucine - Abstract
The integral membrane protein cytochrome f contains an amino-terminal signal sequence that is required for translocation into the thylakoid membrane. The signal sequence contains a hydrophobic core neighbored by an amino-terminal charged residue. Mutations that introduce charged amino acids into the hydrophobic core are inhibitory to cytochrome f translocation, and thus render cells non-photosynthetic. We have isolated both nuclear and chloroplast suppressors of these mutations by selecting for restoration of photosynthetic growth of Chlamydomonas. Here we describe the characterization of two chloroplast, second site suppressor mutations. Both suppressors remove the positively charged amino acid that borders the amino terminus of the hydrophobic core, and replace this arginine with either a cysteine or a leucine. The existence of these suppressors suggests that the hydrophobic core can be shifted in position within the signal sequence, and analysis of triple mutants in the signal confirms this hypothesis. Thus this signal that mediates translocation into the thylakoid membrane is characterized by a hydrophobic region whose exact amino acid content is not critical, and that need not be flanked on its amino terminus by a charged residue.
- Published
- 1996
40. Denitrification, a Novel Type of Respiratory Metabolism in Fungal Mitochondrion
- Author
-
Akiko Takimoto, Sawako Suzuki, Fumiaki Maruo, Yushi Matsuo, Michiyoshi Kobayashi, and Hirofumi Shoun
- Subjects
Nitrite Reductases ,Respiratory chain ,Antimycin A ,Mitochondrion ,Nitrate reductase ,Biochemistry ,Denitrifying bacteria ,chemistry.chemical_compound ,Adenosine Triphosphate ,Oxygen Consumption ,Fusarium ,Nitrate Reductases ,Rotenone ,Guanine Nucleotide Exchange Factors ,Molecular Biology ,Thenoyltrifluoroacetone ,Adaptor Proteins, Signal Transducing ,ATP synthase ,biology ,Proteins ,Cell Biology ,Nitrite reductase ,Mitochondria ,Adaptor Proteins, Vesicular Transport ,Kinetics ,Microscopy, Fluorescence ,Shc Signaling Adaptor Proteins ,chemistry ,Spectrophotometry ,biology.protein ,Cytochromes ,Mitosporic Fungi ,Energy Metabolism - Abstract
Subcellular localization and coupling to ATP synthesis were investigated with respect to the denitrifying systems of two fungi, Fusarium oxysporum and Cylindrocarpon tonkinense. Dissimilatory nitrate reductase of F. oxysporum or nitrite reductase of C. tonkinense could be detected in the mitochondrial fraction prepared from denitrifying cells of each fungus. Fluorescence immunolocalization, cofractionation with mitochondrial marker enzymes, and cytochromes provided evidence that the denitrifying enzymes are co-purified with mitochondria. Respiratory substrates such as malate plus pyruvate, succinate, and formate were effective donors of electrons to these activities in the mitochondrial fractions. Moreover, nitrite and nitrate reduction were shown to be coupled to the synthesis of ATP with energy yields (P:NO3- or P:2e ratios) of 0.88 to 1.4, depending upon whether malate/pyruvate or succinate were provided as substrates. Nitrate or nitrite reductase activity was inhibited by inhibitors such as rotenone, antimycin A, and thenoyltrifluoroacetone. Thus, fungal denitrification activities are localized to mitochondria and are coupled to the synthesis of ATP. The existence of these novel respiration systems are discussed with regard to the origin and evolution of mitochondria.
- Published
- 1996
41. Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli
- Author
-
Yasuhiro Anraku, Keitarou Saiki, Hiroshi Hori, Tatsushi Mogi, and Motonari Tsubaki
- Subjects
Cytoplasm ,Cytochrome ,Stereochemistry ,Molecular Sequence Data ,Ubiquinol oxidase ,Respiratory chain ,Heme ,Biochemistry ,chemistry.chemical_compound ,Bacterial Proteins ,Transferases ,Operon ,Escherichia coli ,Amino Acid Sequence ,Site-directed mutagenesis ,Molecular Biology ,Oxidase test ,Alkyl and Aryl Transferases ,Binding Sites ,biology ,Escherichia coli Proteins ,Spectrum Analysis ,Active site ,Cell Biology ,Cytochrome b Group ,Heme O ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Cytochromes ,Copper - Abstract
The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon. Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach. We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme. Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site. Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.
- Published
- 1993
42. The terminal quinol oxidases of Bacillus subtilis have different energy conservation properties
- Author
-
Mårten Wikström and Marko Lauraeus
- Subjects
Time Factors ,Cytochrome ,Stereochemistry ,Respiratory chain ,Bacillus subtilis ,7. Clean energy ,Biochemistry ,Electron Transport Complex IV ,Cytochrome d Group ,03 medical and health sciences ,Oxygen Consumption ,Tetramethylphenylenediamine ,Cytochrome c oxidase ,Potassium Cyanide ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Oxidase test ,biology ,Cytochrome c ,Cell Membrane ,030302 biochemistry & molecular biology ,Cytochrome d ,NADPH Oxidases ,Cell Biology ,Hydrogen-Ion Concentration ,Cytochrome b Group ,biology.organism_classification ,Kinetics ,Enzyme ,chemistry ,biology.protein ,Cytochromes ,Electrophoresis, Polyacrylamide Gel ,Energy Metabolism ,Oxidoreductases - Abstract
We have analyzed the respiratory chains in the log-arithmic and stationary growth phases of Bacillus subtilis cells grown in rich glucose medium. The cytochrome c branch of the respiratory chain was absent from both types of cells, which used a quinol oxidase branch for respiration. Cytochrome aa3-600 was found to be the major terminal oxidase in log phase cells. This enzyme was shown to translocate protons across the membrane in addition to the charge separation in the oxidation of quinol. Both cytochromes d and aa3-600 were expressed in the stationary phase. After inhibition of the latter by cyanide, cytochrome d was shown to catalyze charge separation during quinol oxidation, but not to pump protons across the membrane. A CO-binding membrane-bound cytochrome of approximately 17 kDa, called cytochrome b558, was presented in log phase cells. This protein did not exhibit oxidase activity and did not have the characteristics of members of the conserved terminal oxidase family.
- Published
- 1993
43. Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth
- Author
-
Kiyoshi Kita, Kiyoshi Konishi, and Yasuhiro Anraku
- Subjects
Ubiquinol ,Cytochrome ,Stereochemistry ,Iron ,Ubiquinol oxidase ,Respiratory chain ,Heme ,Biochemistry ,chemistry.chemical_compound ,Oxygen Consumption ,Multienzyme Complexes ,Escherichia coli ,Cytochrome c oxidase ,Quinone Reductases ,Molecular Biology ,Phospholipids ,Carbon Monoxide ,Oxidase test ,biology ,Escherichia coli Proteins ,Cytochrome d ,Cell Biology ,Cytochrome b Group ,Heme O ,Aerobiosis ,Molecular Weight ,Kinetics ,chemistry ,Spectrophotometry ,biology.protein ,Cytochromes ,Copper - Abstract
Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli K12, was isolated in a highly purified form. The purified oxidase is composed of equimolar amounts of two polypeptides, with Mr = 33,000 and 55,000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 19.5 nmol of heme and 16.8 nmol of copper/mg of protein, but no detectable nonheme iron, phospholipid, ubiquinone, or menaquinone. In the difference spectrum at room temperature, the oxidase shows a single alpha absorption peak at 560 nm and at 77 K it shows two alpha absorption peaks at 555 and 562 nm. This oxidase combines with CO and the CO difference spectrum at room temperature has a peak at 416 nm and a trough at 430 nm in the Soret region. Its oxidation-reduction potential is estimated to be 125 mV (pH 7.4) and it is pH-dependent (-60 mV/pH) in medium of pH 6.0 to 7.4. It catalyzes electron transport to oxygen via ubiquinol and ascorbate in the presence of phenazine methosulfate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity depends on phospholipids and is sensitive to respiratory inhibitors, such as 2-heptyl-4-hydroxyquinoline N-oxide, piericidin A, KCN and NaN3. The divalent cations Zn2+, Cd2+, and Co2+ inhibit the oxidase activity extensively. The oxidase activity of the cytochrome b562-o complex was inhibited by photoinactivation with rose bengal, suggesting that the inhibition by zinc ion results from modification of a histidine residue of cytochrome o.
- Published
- 1984
44. Metabolism of benzo(a)pyrene with isolated hepatocytes and the formation and degradation of DNA-binding derivatives
- Author
-
Bengt Jernström, Sten Orrenius, Helena Vadi, and M D Burke
- Subjects
Male ,Time Factors ,In Vitro Techniques ,Biochemistry ,chemistry.chemical_compound ,Ethers, Cyclic ,Salicylamides ,Hydrocarbons, Chlorinated ,medicine ,Animals ,Benzopyrenes ,Molecular Biology ,Arylsulfatases ,Glucuronidase ,Cell Nucleus ,Flavonoids ,Maleates ,DNA ,Cell Biology ,Metabolism ,Rats ,Cell nucleus ,medicine.anatomical_structure ,Liver ,Benzo(a)pyrene ,chemistry ,Cytochromes ,Degradation (geology) - Published
- 1977
45. Purification and properties of cytochrome b556 in the respiratory chain of aerobically grown Escherichia coli K12
- Author
-
Kiyoshi Kita, I Yamato, and Yasuhiro Anraku
- Subjects
Cytochrome ,Iron ,Size-exclusion chromatography ,Respiratory chain ,Heme ,Biochemistry ,chemistry.chemical_compound ,Escherichia coli ,Amino Acids ,Sodium dodecyl sulfate ,Molecular Biology ,Gel electrophoresis ,Chromatography ,biology ,Cell Biology ,Cytochrome b Group ,Aerobiosis ,Isoelectric point ,chemistry ,Spectrophotometry ,Sephadex ,biology.protein ,Cytochromes ,Oxidation-Reduction - Abstract
Cytochrome b556, a major component of type b cytochromes in the respiratory chain of aerobically grown Escherichia coli, was purified to near homogeneity. It was solubilized from cytoplasmic membranes by treatment with Sarkosyl/cholate mixture and purified by gel filtration on Sephadex G-200. The purified cytochrome b556 is an oligomer composed of identical polypeptides, with a molecular weight of 17,500, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains equimolar amounts of heme and polypeptide but no detectable non-heme iron, phospholipid, or dehydrogenase. Its isoelectric point was determined to be 8.5. The cytochrome b556 is highly hydrophobic in its amino acid composition and does not contain any half-cystine residues. The purified cytochrome b556 is spectrophotometrically pure and the alpha absorption peak in its difference spectrum at 77 K is at 556 nm. The molar extinction coefficient of cytochrome b556 was determined as 22.8 cm-1 mM-1. Its oxidation-reduction potential was found to be -45 mV. It could be reduced by D-lactate dehydrogenase of E. coli in the presence of menadione.
- Published
- 1978
46. The Pigment Complement of the Photosynthetic Reaction Center Isolated from Rhodospirillum rubrum
- Author
-
Gabriel Gingras and Michel van der Rest
- Subjects
Chlorophyll ,Photosynthetic reaction centre ,Time Factors ,Light ,Photochemistry ,Ultrafiltration ,Rhodospirillum rubrum ,Photosynthesis ,Biochemistry ,Light-harvesting complex ,Pigment ,chemistry.chemical_compound ,Molecular Biology ,biology ,Chemistry ,Bacterial Chromatophores ,Pigments, Biological ,Cell Biology ,Molar absorptivity ,biology.organism_classification ,Kinetics ,Spectrophotometry ,Extinction (optical mineralogy) ,visual_art ,Mutation ,visual_art.visual_art_medium ,Cytochromes ,Spectrophotometry, Ultraviolet ,Chromatography, Thin Layer ,Bacteriochlorophyll ,Oxidation-Reduction ,Mathematics - Abstract
Isolated photosynthetic reaction center from the bacterium Rhodospirillum rubrum was extracted with acetone-methanol. Its main pigments were identified as bacteriochlorophyll, bacteriopheophytin, and spirilloxanthin. The extinction coefficients of these pigments in acetone-methanol were determined. Quantitative spectroscopic analysis of the dry acetone-methanol extracts indicated a bacteriochlorophyll to bacteriopheophytin mole ratio of 2 and led to an extinction coefficient at 868 nm of 71.3, 142.6, or 214 mm-1 cm-1, depending on whether the photosynthetic center was assumed to contain 2, 4, or 6 bacteriochlorophyll molecules. The extinction coefficient of P870 was determined by mixing photo-oxidized photosynthetic center with ferrocytochrome c in a stopped flow spectrophotometer. Complete reaction was observed when the reactants were mixed in equimolar ratio calculated on the basis of an extinction coefficient of 143 mm-1 cm-1 at 868 nm. This photosynthetic center is proposed to contain 4 moles of bacteriochlorophyll, 2 moles of bacteriopheophytin, and 1 mole of spirilloxanthin per equivalent of P870.
- Published
- 1974
47. Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2
- Author
-
Claude Balny, Patrick Maurel, and Claude Bonfils
- Subjects
Cytochrome ,Stereochemistry ,Photochemistry ,Biochemistry ,Electron Transport ,Hydroxylation ,chemistry.chemical_compound ,Electron transfer ,Cytochrome P-450 Enzyme System ,Cytochrome b5 ,medicine ,Animals ,Molecular Biology ,Micelles ,biology ,Cell Biology ,Electron transport chain ,Molecular Weight ,Dissociation constant ,Kinetics ,Cytochromes b5 ,chemistry ,Spectrophotometry ,Phenobarbital ,Microsomes, Liver ,biology.protein ,Cytochromes ,Ferric ,Rabbits ,Benzphetamine ,Oxidation-Reduction ,medicine.drug - Abstract
Interaction and electron transfer between highly purified microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 from uninduced rabbits was investigated by difference and stopped-flow spectrophotometry. Formation of a 1:1 complex between ferric P-450 and b5, demonstrated by difference spectrophotometry, was observed only when both cytochromes were incorporated into micelles of phosphatidylcholine. The dissociation constant (Kd) of the complex was decreased from 2.3 microM to 0.4 microM in the presence of 1 mM benzphetamine. The apparent Kd of benzphetamine was reduced from 220 microM to 50 microM upon addition of b5. The influence of ferrous b5 on the autooxidation of the oxyferrous intermediate of P-450 in the presence and in the absence of substrate was investigated by stopped-flow spectrophotometry. Both cytochromes were reduced photochemically, so that experiments could be carried out in the absence of the corresponding reductases and reduced pyridine nucleotides. Kinetic analysis of the data showed that formation of a 1:1 complex between ferrous P-450 and b5 was a prerequisite for electron transfer between the cytochromes. Here again, incorporation of both cytochromes into micelles was absolutely required for this process. Kd was decreased from 7.5 microM to 2.2 microM in the presence of 1 mM benzphetamine. The rate of electron transfer from b5 to oxyferrous P-450, derived from the kinetics of reoxidation of either b5 or P-450, was increased from 2.5 s-1 to about 4 to 7 s-1 in the presence of 1 mM benzphetamine. These results provide the first quantitative data on the electron transfer between the b5 and P-450, whose rate constant is compatible with the observations made on the effect of b5 on hydroxylation reactions catalyzed by the P-450 enzyme system.
- Published
- 1981
48. Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria
- Author
-
Efraim Racker, B I Kanner, and Ramón Serrano
- Subjects
Oligomycin ,ATPase ,Cytochrome c Group ,Cytochromes c1 ,Oxidative phosphorylation ,Biochemistry ,chemistry.chemical_compound ,Adenine nucleotide ,Animals ,Cytochrome c oxidase ,NADH, NADPH Oxidoreductases ,Submitochondrial particle ,Molecular Biology ,Phospholipids ,Adenosine Triphosphatases ,biology ,Chemistry ,Myocardium ,Vesicle ,ATPase complex ,Cell Biology ,Mitochondria, Muscle ,Succinate Dehydrogenase ,biology.protein ,Cytochromes ,Cattle ,Mitochondrial ADP, ATP Translocases - Abstract
1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3.The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.
- Published
- 1976
49. Requirements for unsaturated fatty acids for the induction on respiration in Saccharomyces cerevisiae
- Author
-
RW Walenga and WE Lands
- Subjects
Oleic Acids ,Saccharomyces cerevisiae ,Cycloheximide ,Biology ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Oxygen Consumption ,Eicosanoic Acids ,Respiration ,Protein biosynthesis ,Glycerol ,Molecular Biology ,Derepression ,Unsaturated fatty acid ,chemistry.chemical_classification ,fungi ,Fatty acid ,Cell Biology ,Kinetics ,Oleic acid ,Chloramphenicol ,chemistry ,Fatty Acids, Unsaturated ,Cytochromes - Abstract
Unsaturated fatty acids provided during the release from glucose repression were shown to be essential for derepression of respiration in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Cells derepressed in the presence of oleic acid contained three to six times as much cytochrome per cell as those derepressed in the absence of unsaturated fatty acid or those derepressed with eicosaenoic acid. The delta9 isomer was the most efficient of the cis-octadecenoic acid isomers in supporting that increase, and eicosaenoic acid supported an increase at only 15% the rate observed with oleic acid. Derepression, even in the presence of oleic acid, proceeded only after a lag of 3 hours. When glucose was removed prior to the addition of oleate, the lag was reduced by the time of the preincubation with glycerol. This result suggests that some processes necessary for increased respiration can proceed in the absence of an added unsaturated fatty acid, but these processes apparently require certain levels of unsaturated acids in the pre-existing lipids, since they occurred in cells whose membranes contained 50 mol % oleate, but not in cells containing only 20 mol %. These processes leading to eventual increased respiration were inhibited by cycloheximide but not chloramphenicol, suggesting that protein synthesis on cytoplasmic ribosomes but not mitochondrial ribosomes were required. Derepression in the absence of oleate for 3 hours lessened the inhibition or respiration induction by ethidium bromide. This result indicates that the transcription of mitochondrial DNA necessary for the induction of respiration may have occurred in the absence of added unsaturated fatty acid, but that some subsequent event required added esterified unsaturated fatty acid.
- Published
- 1975
50. The Amino Acid Sequence of Cytochrome b5
- Author
-
Juris Ozols and Philipp Strittmatter
- Subjects
Biochemistry ,Hydrolysis ,Cytochrome b5 ,medicine ,Animals ,Chymotrypsin ,Trypsin ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,biology ,Chemistry ,Cell Biology ,Chromatography, Ion Exchange ,Amino acid ,Liver ,Amino acid composition ,Chromatography, Gel ,biology.protein ,Microsome ,Cytochromes ,Cattle ,medicine.drug - Abstract
Calf liver microsomal apocytochrome b5 was hydrolyzed with chymotrypsin and the resulting peptides were resolved on a Dowex 1 column. The isolation and partial amino acid sequences of the 10 chymotryptic peptides are described. The total amino acid composition of these peptides is equal to the sum of the residues present in the tryptic peptides. On the basis of the data obtained for the chymotryptic peptides the order of the tryptic peptides was established. This information, combined with the known sequences of the tryptic peptides, provided sufficient evidence to construct a unique amino acid sequence for the 85 amino acid residues in cytochrome b5.
- Published
- 1968
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