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At least 757 records · Page 42Linked to original sources
PubMed1995

Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase.

The structure of pseudoazurin from Thiosphaera pantotropha has been determined and compared to structures of both soluble and membrane-bound periplasmic redox proteins. The results show a matching set of unipolar, but promiscuous, docking motifs based on a positive hydrophobic surface patch on the electron shuttle proteins pseudoazurin and cytochrome c550 and a negative hydrophobic patch on the surface of their known redox partners. The observed electrostatic handedness is argued to be associated with the charge-asymmetry of the membrane-bound components of the redox chain due to von Heijne's 'positives-inside' principle. We propose a 'positives-in-between' rule for electron shuttle proteins, and expect a negative hydrophobic patch to be present on both the highest and lowest redox potential species in a series of electron carriers.

Azurin↗
PubMed2006

Metalloprotein tunnel junctions: compressional modulation of barrier height and transport mechanism.

Though the incorporation of sensory or potentially-switchable biological entities into electronic devices brings with it a number of complicating issues associated with hydration, structural complexity/delicacy, and low conductance, the possibility of resolving properties of fundamental importance (such as the influence of protein fold on conductance) at a molecularly-resolved level, are exciting. Our ability to analyse charge transport through a biological macromolecule remains, though, a significant practical and theoretical challenge. Though much information can be gained by carrying out such examinations at a molecular level, there exist few methods where such controlled analyses are, in fact, feasible. Here we report on the electron transport characteristics of a blue copper metalloprotein as characterized by conductive-probe atomic force microscopy. At very low imposed force, contact resistance is high, electrical contact unstable, and the junction undergoes dielectric breakdown at 1.1-1.5 GV m(-1). At increased applied force, the current-voltage characteristics are entirely reproducible and well-described by a Simmons (non-resonant) tunnelling model. Though highly resistive, observations demonstrate the ability of the protein matrix to mediate appreciable tunnelling current. Non-resonant behaviour is consistent with observations of bias-independent tunnelling imaging. In fitting observed transport characteristics to this model, it is possible to deconvolute barrier height and length at specific experimental conditions and, specifically, to monitor the modulation of these parameters by imposed compressional force. At higher field spectroscopic features assignable to metal based density of states are reproducibly observed. These vanish in a force regime where the tunnel barrier to direct tip-sample communication decreases.

Azurin↗
PubMed1979

The mechanism of reduction of single-site redox proteins by ascorbic acid.

The reduction of single-site haem and copper redox proteins by ascorbic acid was studied as a function of pH. Evidence is presented that indicates that the double-deprotonated ascorbate anion, ascorbate2-, is the reducing agent, and the pH-independent second-order rate constants for reduction by this species are given. Investigation of the temperature dependences of these rate constants have yielded the values of the activation parameters (delta H++ and delta S++) for reduction. These values, together with ligand-replacement studies, suggest that ascorbate2- acts as an outer-sphere reductant for these proteins. Reasons to account for the apparent inability of ascorbic acid to reduce the alkaline conformer of mammalian ferricytochrome c are suggested.

Ascorbic Acid↗
PubMed1992

A quantitative model for the mechanism of action of the cytochrome c peroxidase of Pseudomonas aeruginosa.

Each of the elementary reaction steps in both the activation process and catalytic cycle of the cytochrome c peroxidase of Pseudomonas aeruginosa was characterized using stopped-flow methods. A synthesis of these data led to the establishment of a quantitative model for the action of this enzyme. Comparisons were made between experimental data and calculations over a wide range of enzyme, reductant and H2O2 concentrations. Close agreement was found between empirical and simulated reaction time courses from millisecond to tens of seconds time ranges, giving us confidence in the validity of the quantitative model of this enzyme's actions.

Azurin↗
PubMed1995

The complete amino acid sequence confirms the presence of pseudoazurin in Thiosphaera pantotropha.

The complete amino acid sequence, obtained by direct protein sequencing, of the pseudoazurin from Thiosphaera pantotropha is reported. It shows sequence identities varying from 46 to 66% with previously sequenced pseudoazurins. Previously identified conserved residues with key functions in pseudoazurins are found in the protein from T. pantotropha with the exception of glycine-39, the carbonyl group of which has been considered as a ligand to the copper, which is replaced by a serine residue. Electrospray-ionization MS (ESI-MS) has shown that pseudoazurin from T. pantotropha often contains two polypeptide species differing in molecular mass by 16 Da, presumably owing to oxidation of a methionine residue to a sulphoxide derivative. These two species have different endoproteinase Arg-C digestion patterns. Conditions for ESI-MS were identified that permitted either the retention or the loss of the single copper ion associated with the pseudoazurin. The aberrant tendency of T. pantotropha pseudoazurin to form a disulphide-bridged dimer on SDS/PAGE under some conditions is described.

Amino Acid Sequence↗
PubMed1997

The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli.

The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to approximately 80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.

Amino Acid Sequence↗
PubMed2000

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Animals↗
PubMed2003

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes.

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.

Amino Acid Sequence↗
PubMed1976

Applications of synchrotron radiation to protein crystallography: preliminary results.

X-ray diffraction photographs of protein single crystals have been obtained using synchrotron radiation produced by an electron-positron storage ring. The diffracted intensities observed with this unconventional source are a factor of at least 60 greater than those obtained with a sealed x-ray tube using the same crystal and instrumental parameters. Diffraction data have been collected by the precession method to higher resolution and using smaller protein crystals than would have been possible with a conventional source. The crystal decay rate in the synchrotron beam for several proteins appears to be substantially less than that observed with Ni-filtered Cu radiation. The tunable nature of the source (which allows selective optimization of anomalous contributions to the scattering factors) and the low angular divergence of the beam make the source very useful for single crystal protein diffraction studies.

Asparaginase↗
PubMed1989

Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.

Amino Acid Sequence↗
PubMed1995

Spectroscopic and electrochemical studies on active-site transitions of the type 1 copper protein pseudoazurin from Achromobacter cycloclastes.

The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode. Measurements carried out at 25.0 degrees C indicate a midpoint reduction potential of E 1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of delta Ep = 59 mV. The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 x 10(-6) cm2 s-1 and 3.7 x 10(-2) cm s-1, respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple. The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 x 10(-3) cm s-1, consistent with a slower process at the positively charged electrode surface. At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure. The trigonal form has elongated axial bonding and an axial EPR spectrum. At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm-1 are shifted to higher energy (approximately 10 cm-1), consistent with a stronger Cu-S bond.

Alcaligenes↗
PubMed1995

The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with NO2- bound and with type II copper depleted.

High resolution x-ray crystallographic structures of nitrite reductase from Achromobacter cycloclastes, undertaken in order to understand the pH optimum of the reaction with nitrite, show that at pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other than in the occupancy of the type II copper at the active site. An extensive network of hydrogen bonds, both within and between subunits of the trimer, maintains the rigidity of the protein structure. A water occupies a site approximately 1.5 A from the site of the type II copper in the structure of the type II copper-depleted structure (at pH 5.4), again with no other significant changes in structure. In nitrite-soaked crystals, nitrite binds via its oxygens to the type II copper and replaces the water normally bound to the type II copper. The active-site cavity of the protein is distinctly hydrophobic on one side and hydrophilic on the other, providing a possible path for diffusion of the product NO. Asp-98 exhibits thermal parameter values higher than its surroundings, suggesting a role in shuttling the two protons necessary for the overall reaction. The strong structural homology with cupredoxins is described.

Alcaligenes↗
PubMed1996

Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6.

Site-directed mutagenesis of a copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to identify the amino acid residues involved in interaction with its redox partner, pseudoazurin, in which four positively charged residues were previously shown to be important in the interaction. Ten negatively charged residues located on the surface of NIR were replaced independently by alanine or serine. All the altered NIRs showed CD spectra and optical spectra identical to those of wild-type NIR, suggesting that all the replacements caused no gross change in the overall structure or in the environment of type 1 copper site. Kinetic analysis of electron transfer between pseudoazurin and altered NIRs revealed that the replacement of Glu-118, Glu-197, Asp-201, Glu-204, or Asp-205 by Ala caused a significant increase in the Km value for pseudoazurin compared with that of wild-type NIR. Furthermore, the simultaneous replacement of three of these residues (Glu-118, Glu-197, and Asp-201) caused a further increase in the Km value. These results suggested that the negatively charged residues are involved in electrostatic interaction with pseudoazurin. Kinetic analyses of the altered NIRs (E118A, E197A, or D201A) with altered pseudoazurins (K10A, K57A, or K77A) implicate specific pairs of the charged residues that are involved in electrostatic interaction between NIR and pseudoazurin.

Alcaligenes↗
PubMed1999

Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes. Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine.

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state. A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.

Alcaligenes↗
PubMed1999

Interaction-induced redox switch in the electron transfer complex rusticyanin-cytochrome c(4).

The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidans displays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the epsilon-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c(4), induced a decrease of rusticyanin's redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers.

Acidithiobacillus thiooxidans↗
PubMed2004

Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin.

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k(ET) values of these mutants become progressively larger as the number of mutated residues increases. The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.

Achromobacter denitrificans↗
PubMed2005

Transient homodimer interactions studied using the electron self-exchange reaction.

Transient homodimer protein interactions have been investigated by analyzing the influence of ionic strength (NaCl) on the electron self-exchange (the bimolecular reaction whereby the two oxidation states of a redox protein interconvert) rate constant (k(ese)) of four plastocyanins. The k(ese) values for the plastocyanins from spinach, Dryopteris crassirhizoma (a fern), and the green alga Ulva pertusa, which possess acidic patches of varying size and locations, increase 190-, 29-, and 21-fold, respectively, at elevated ionic strength (I = 2.03 M). In contrast, the k(ese) for the almost neutral cyanobacterial plastocyanin from Anabaena variabilis exhibits very little dependence on ionic strength. The temperature dependence of the k(ese) for spinach plastocyanin (I = 0.28 M) provides evidence for poor packing at the homodimer interface. Representative structures of the transient homodimers involved in electron self-exchange, which are consistent with fits of the ionic strength dependence of k(ese) to van Leeuwen theory, have been obtained from protein modeling and docking simulations. The Coulombic energy of the docked homodimers follows the order spinach > D. crassirhizoma > U. pertusa > A. variabilis, which matches that of the overall influence of ionic strength on k(ese). Analysis of the homodimer structures indicates that poor packing and high planarity are features of the interface that favor transient interactions. The physiologically relevant Mg2+ ion has a much more pronounced influence on the k(ese) of spinach plastocyanin, which along with the known properties of the thylakoid lumen suggests a biological role for electron self-exchange.

Amino Acid Sequence↗