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PubMed1982

Model of the active site in the blue oxidases based on the ceruloplasmin-plastocyanin homology.

Available sequence information (a total of 650 residues out of about 1,050) of human ceruloplasmin, a blue copper-containing oxidase, has been examined for internal homologies and relationships to other blue proteins. The peptide chain has an evident 3-fold repeat of about 340 residues, and weak evidence for a 6-fold repeat of 170 residues exists. When another method was used to compare the longer sequence with the sequences of small blue proteins, azurins and plastocyanins, a 109-residue-long sequence at the COOH terminus of ceruloplasmin was found to be homologous to the plastocyanins. The alignment obtained was used to construct, on a graphic display, a three-dimensional model of this part of ceruloplasmin by using the coordinates for popular plastocyanin. Deletions and insertions could be accommodated in turns and kinks in the essentially eight-stranded pleated sheet molecule wherein each of the hydrophobic core residues was conserved or conservatively replaced. Eight of the 12 histidine side chains were clustered at or close to the binding site for the blue (type 1) copper. On the assumption that these are copper ligands, a model for the active site of ceruloplasmin containing four copper ions could be constructed in a manner consistent with known spectroscopic and kinetic data. In particular, two of the coppers are close enough (3 A) to form a binuclear center. The positions of the two additional coppers (the fifth and the sixth) in ceruloplasmin are suggested on the basis of the internal homologies.

Amino Acid Sequence↗
PubMed1997

Effect of protein dynamics on biological electron transfer.

Computer simulations of the effect of protein dynamics on the long distance tunneling mediated by the protein matrix have been carried out for a Ru-modified (His 126) azurin molecule. We find that the tunneling matrix element is a sensitive function of the atomic configuration of the part of the protein matrix in which tunneling currents (pathways) are localized. Molecular dynamics simulations show that fluctuations of the matrix element can occur on a time scale as short as 10 fs. These short time fluctuations are an indication of a strong dynamic coupling of a tunneling electron to vibrational motions of the protein nuclear coordinates. The latter results in a modification of the conventional Marcus picture of electron transfer in proteins. The new element in the modified theory is that the tunneling electron is capable of emitting or absorbing vibrational energy (phonons) from the medium. As a result, some biological reactions may occur in an activationless fashion. An analytical theoretical model is proposed to account for thermal fluctuations of the medium in long distance electron transfer reactions. The model shows that, at long distances, the phonon-modified inelastic tunneling always dominates over the conventional elastic tunneling.

Computer Simulation↗
PubMed1995

The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum.

A copper-containing nitrite reductase (Cu-NiR) was purified to homogeneity from the denitrifying fungus Fusarium oxysporum. The enzyme seemed to consist of two subunits with almost the same M(r) value of 41,800 and contains two atoms of copper per subunit. The electron paramagnetic resonance spectrum showed that both type 1 and type 2 copper centers are present in the protein, whereas the visible absorption spectrum exhibited a sole and strong absorption maximum at 595 nm, causing a blue but not green color. The reaction product due to the Cu-NiR was mainly nitric oxide (NO), whereas a stoichiometric amount of nitrous oxide (N2O) was formed when cytochrome P-450nor was further added to the assay system. Therefore, the denitrifying (N2O forming) nitrite reductase activity that we had detected in the cell-free extract of the denitrifying cells (Shoun, H., and Tanimoto, T. (1991) J. Biol. Chem. 266, 11078-11082) could be reconstituted upon combination of the purified Cu-NiR and P-450nor. The Km for nitrite and specific activity at pH 7.0 were estimated as 49 microM and 447 mumol NO.min-1.mg protein-1, respectively. Its activity was strongly inhibited by cyanide, carbon monoxide, and diethyldithiocarbamate, whereas enormously restored by the addition of cupric ions. An azurin-like blue copper protein (M(r) = 15,000) and a cytochrome c were also isolated from the same fungus, both of which together with cytochrome c of the yeast Saccharomyces cerevisiae were effective in donating electrons to the fungal Cu-NiR. The result suggested that the physiological electron donor of the Cu-NiR is the respiratory electron transport system. The intracellular localization of Cu-NiR was investigated, and it was suggested that the Cu-NiR localizes in an organelle such as mitochondrion. These findings showed the identity in many aspects between the fungal nitrite reductase and bacterial dissimilatory Cu-NiRs. This is the first isolation of dissimilatory NiR from a eukaryote.

Bacterial Proteins↗
PubMed1999

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Amino Acid Sequence↗
PubMed1992

Towards an automatic method of predicting protein structure by homology: an evaluation of suboptimal sequence alignments.

A major problem in predicting protein structure by homology modelling is that the sequence alignment from which the model is built may not be the best one in terms of the correct equivalencing of residues assessed by structural or functional criteria. A useful strategy is to generate and examine a number of suboptimal alignments as better alignments can often be found away from the optimal. A procedure to filter rapidly suboptimal alignments based on measurement of core volumes and packing pair potentials is investigated. The approach is benchmarked on three pairs of sequences which are non-trivial to align correctly, namely two immunoglobulin domains, plastocyanin with azurin and two distant globin sequences. It is shown to be useful to reduce a large ensemble of possible alignments down to a few which correspond more closely to the correct (structure based) alignment.

Algorithms↗
PubMed1987

Electron-transfer biosensors.

The electrochemistry of redox proteins is now well established. Conditions exist which allow electron-transfer reactions of all simple proteins to proceed rapidly and reversibly at electrodes. Coupling of the electrode reaction to enzymes, for which the redox proteins act as cofactors, allows exploitation of this good electrochemistry. This is well illustrated by the enzyme-catalysed electrochemical oxidation of p-cresol to p-hydroxybenzaldehyde, which has been shown to proceed along with coupling to the electrode via the copper protein, azurin, or the organometallic compound ferroceneboronic acid. Ferrocene derivatives, in general, show a degree of versatility, coupling the electron-transfer reactions of many enzymes. Thus derivatives of the ferricinium ion act as excellent electron-transfer reagents from the enzyme glucose oxidase. The system is capable of detecting glucose in blood. Similar procedures, in conjunction with the appropriate enzyme, have yielded assays for, among others, H2O2 and cholesterol.

Biotechnology↗
PubMed2004

Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151.

Dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151 (HdNIR) is a novel copper-containing nitrite reductase (CuNIR) composed of six identical subunits. One plastocyanin-like domain and one green CuNIR-like domain are connected to each other, suggesting that the HdNIR subunit structure resembles a complex of green CuNIR and pseudoazurin (or azurin). Recombinant HdNIR protein was crystallized using the hanging-drop vapour-diffusion method with PEG 4000 as the precipitant at pH 8.9. X-ray diffraction data were collected to 2.35 A resolution. The HdNIR crystal belonged to the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = b = 221.9, c = 165.2 A, giving 12 molecules (two hexamers) per asymmetric unit and a solvent content of 64%. A mutant form of HdNIR, C260A, which lacks the type I copper ion in the CuNIR-like domain, was prepared and crystallized under wild-type HdNIR conditions. The C260A mutant crystal belonged to the cubic space group P4(3)32 (or P4(1)32), with unit-cell parameters a = b = c = 153.7 A, giving one molecule per asymmetric unit and a solvent content of 59%. X-ray diffraction data were collected to 3.5 A resolution. To solve the crystal structure of HdNIR, the multiwavelength anomalous dispersion (MAD) method and the molecular-replacement method are currently being used.

Crystallization↗
PubMed2000

Blue copper proteins: a comparative analysis of their molecular interaction properties.

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

Amino Acid Sequence↗
PubMed1998

UV-difference and CD spectroscopy studies on juvenile hormone binding to its carrier protein.

Juvenile hormone binding protein (JHBP) from hemolymph of Galleria mellonella is a single-chain glycoprotein of molecular mass near 25,880 containing no Trp residues. The fourth derivative of the protein absorption spectrum shows the characteristic vibrational components of the phenylalanine spectrum within the range 240-270 nm. At longer wavelengths two main bands 0-0 and 0+800 cm(-1) appear, caused by vibrational levels of electronic transition, pi-->pi*, in the tyrosine residues, with maxima at 279 nm and 286 nm, respectively. Two intersection points of the second derivative absorption band with abscissa at about 288.8 nm and 283 nm were analysed for estimation of the environment polarity of Tyr residues in the JHBP molecule. The results obtained suggest that JHBP contains at least two classes of Tyr residues with very apolar environment, similar to that found in azurine. In the JHBP-JH complex only one class of Tyr residues located in a very apolar environment was found, and a small perturbation of disulphide bridges was deduced from the UV-difference spectrum. Ligand perturbation appears as a minimum at 243 nm of the UV-difference spectrum. Comparison of the circular dichroism (CD) spectra for free JHBP with the CD spectra for the JHBP-JH complex monitored in the far-UV (190-240 nm) region indicates rather small differences in the secondary structure of the protein. Although ligand binding induces distinct changes in the near-UV (250-300 nm) region of the CD spectrum of JHBP, it is apparent where both Tyr and Phe residues contribute.

Animals↗
PubMed1979

Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization.

1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.

Amino Acids↗
PubMed1981

Spectroscopic and photoreduction studies of copper chromophores in ceruloplasmin.

Photoreduction of native ceruloplasmin, using the 454.5-nm line of an Ar+ laser, enables the identification of type-Ia, type-Ib and type-II copper. The circular dichroic spectra of N--3-bound type-II copper and SCN-- -bound type-II copper are obtained by the same procedure after anionic treatment of ceruloplasmin. From circular dichroic and resonance Raman evidence it appears that some of type-Ia and type-Ib copper ligands differ. Type-Ib copper ligands seem to the same as type-I copper in plastocyanin and azurin. Even though type-Ib copper is coordinated to one sulfur of cysteine and one sulfur of methionine (or disulfide of cystine), the methionine sulfur is not a ligand for type-Ia copper.

Azides↗
PubMed1983

Identification of the thiol groups in human ceruloplasmin.

Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from five different areas in the molecule and together accounted for 92 residues, six of which were cysteines. Since one of the peptides contained two cysteines it seemed evident that, prior to the reductive elution of the peptides, one of these had been paired in a disulfide bridge with one of the four remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The second (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the approximately 1050 residues present in the protein. Three of them also show about 40% identity with each other, whereas no relatedness is observed with the fourth. The three related peptides are, moreover, clearly homologous to the copper-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the two sequences reported for the blue oxidase, fungal laccase, one of which contains a disulfide bridge.

Amino Acid Sequence↗
PubMed1986

Complete amino acid sequence of plastocyanin from a green alga, Enteromorpha prolifera.

The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.

Amino Acid Sequence↗
PubMed2005

Bifidobacterium longum endogalactanase liberates galactotriose from type I galactans.

A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37 degrees C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The K(m) and V(max) for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (beta1-->4)galactans and (beta1-->4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (beta1-->3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

Amino Acid Sequence↗
PubMed2006

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Bacterial Proteins↗
PubMed1996

Electron transfer in proteins.

Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization energy (lambda) and the electronic-coupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed lambda and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: beta sheets appear to mediate coupling more efficiently than alpha-helical structures, and hydrogen bonds play a critical role in both.

Electrons↗
PubMed2004

Spin distribution and the location of protons in paramagnetic proteins.

Two current frontiers in EPR research are high-field ( nu0 > 70 GHz, B0 > 2.5 T ) electron paramagnetic resonance (EPR) and high-field electron-nuclear double resonance (ENDOR). This review focuses on recent advances in high-field ENDOR and its applications to the study of proteins containing native paramagnetic sites. It concentrates on two aspects; the first concerns the determination of the location of protons and is related to the site geometry, and the second focuses on the spin density distribution within the site, which is inherent to the electronic structure. Both spin density and proton locations can be derived from ligand hyperfine couplings determined by ENDOR measurements. A brief description of the experimental methods is presented along with a discussion of the advantages and disadvantages of high-field ENDOR compared with conventional X-band (~ 9.5 GHz) experiments. Specific examples of both protein single crystals and frozen solutions are then presented. These include the determination of the coordinates of water ligand protons in the Mn(II) site of concanavalin A, the detection of hydrogen bonds in a quinone radical in the bacterial photosynthetic reaction center as well as in the tyrosyl radical in ribonuclease reductase, and the study of the spin distribution in copper proteins. The copper proteins discussed are the type I copper of azurin and the binuclear CuA center in a number of proteins. The last part of the review presents a brief discussion of the interpretation of hyperfine couplings using quantum chemical calculations, primarily density functional theory (DFT) methods. Such methods are becoming an integral part of the data analysis tools, as they can facilitate signal assignment and provide the ultimate relation between the experimental hyperfine couplings and the electronic wave function.

Binding Sites↗
PubMedDate not supplied

Bone aluminium content in Alzheimer's disease.

The possible association between aluminium and Alzheimer's disease is still contentious. If aluminium neurotoxicity is implicated in the pathophysiology of Alzheimer's disease, it may result either from excessive aluminium exposure or increased brain aluminium uptake. In a pilot study to test the former hypothesis, trabecular bone aluminium content, which reflects long-term aluminium exposure, was evaluated in 7 patients with a clinical diagnosis of senile dementia of the Alzheimer type (SDAT; mean age 80.8 +/- 3.35 years) and 19 non-demented age-matched controls (mean age 79.6 +/- 6.09 years). Trabecular bone was obtained from post-traumatic femoral neck fracture specimens taken from patients during femoral head prosthesis surgery. Bone aluminium content was expressed quantitatively by atomic absorption spectrometry and qualitatively by the acid solochrome azurine histological staining technique. Quantitative analysis showed a lower aluminium content in the SDAT (11.9 +/- 4.04 micrograms/g dry bone) versus the non-demented group (18.2 +/- 7.37 micrograms/g), which was significant at the 95% but not at the 99% confidence limit. Aluminium deposition from qualitative histological analysis was not detectable in either group. These results do not support a hypothesis of excessive aluminium absorption and tissue accumulation in Alzheimer's disease.

Age Distribution↗