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1.
The 1H-NMR spectrum of cucumber basic blue protein (CBP) has been recorded. Examination of the spectrum of the reduced protein suggests that one or more sidechains exist in conformations which interconvert slowly at ambient temperatures. His 39, His 84 and Met 89 are identified as copper ligands by redox titration and by amino acid sequence homology with plastocyanin and azurin. The importance of a Phe sidechain close to the Met ligand in the potential blue copper site is confirmed. Broadening of His ligand resonances at elevated temperatures reveals an exchange process at the reduced copper centre. 相似文献
2.
Munime Lundeen 《Journal of inorganic biochemistry》1985,25(4):277-291
The frequencies of occurrence of the side chains in proteins in the first, second, third, and fourth positions of a reverse turn in a set of 26 nonredundant protein chains are shown in a table that lists cysteine and cystine side chains separately. This table was used to predict the reverse turns in poplar plastocyanin whose crystal structure is known (75% of the turn residues are correctly predicted but the overall accuracy of the predictions is only 66% in a turn-not-turn two-state model), and in three blue copper proteins whose crystal structures are being determined (cucumber plastocyanin and cucumber basic protein) or contemplated (Rhus vernificera stellacyanin). The copper cores proposed for cucumber basic protein and stellacyanin are discussed. 相似文献
3.
A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 A resolution. 总被引:1,自引:1,他引:0
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P. J. Hart A. M. Nersissian R. G. Herrmann R. M. Nalbandyan J. S. Valentine D. Eisenberg 《Protein science : a publication of the Protein Society》1996,5(11):2175-2183
Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed. 相似文献
4.
A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model. 相似文献
5.
Xie Y Inoue T Miyamoto Y Matsumura H Kataoka K Yamaguchi K Nojini M Suzuki S Kai Y 《Journal of biochemistry》2005,137(4):455-461
Mavicyanin, a glycosylated protein isolated from Cucurbita pepo medullosa (zucchini), is a member of the phytocyanin subfamily containing one polypeptide chain of 109 amino residues and an unusual type-I Cu site in which the copper ligands are His45, Cys86, His91, and Gln96. The crystal structures of oxidized and reduced mavicyanin were determined at 1.6 and 1.9 A resolution, respectively. Mavicyanin has a core structure of seven polypeptide beta-strands arranged as a beta-sandwich organized into two beta-sheets, and the structure considerably resembles that of stellacyanin from cucumber (CST) or cucumber basic protein (CBP). A flexible region was not observed on superimpositioning of the oxidized and reduced mavicyanin structures. However, the Cu(II)-epsilon-O-Gln96 bond length was extended by 0.47 A, and the Thr15 residue was rotated by 60.0 degrees and O-gamma1-Thr15 moved from a distance of 4.78 to 2.58 A from the ligand Gln96 forming a new hydrogen bond between O-gamma1-Thr15 and epsilon-O-Gln96 upon reduction. The reorganization of copper coordination geometry of mavicyanin upon reduction arouses reduction potential decreased above pH 8 [Battistuzzi et al. (2001) J. Inorg. Biochem. 83, 223-227]. The rotation of Thr15 and the hydrogen bonding with the ligand Gln96 may constitute structural evidence of the decrease in the reduction potential at high pH. 相似文献
6.
Gunnar Fröman 《FEBS letters》1982,143(2):220-224
Absorption, circular dichroism, electron spin resonance and resonance Raman spectra of a blue copper protein, plantacyanin from cucumber peel have been measured and these spectral properties compared with those of other blue copper proteins. From the spectral properties, amino acid analysis and redox potential, we discuss the active site and redox properties of this protein. 相似文献
7.
Gianantonio Battistuzzi Marco Borsari Lodovica Loschi M. Sola 《Journal of biological inorganic chemistry》1997,2(3):350-359
The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques
in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include
a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced
protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to
some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared
with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations.
This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein
class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution
to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N2S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties
and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations
in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts
for the ionic strength dependence of the reduction potential.
Received: 20 December 1996 / Accepted: 26 March 1997 相似文献
8.
Davidson VL 《Archives of biochemistry and biophysics》2004,428(1):32-40
Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions. 相似文献
9.
Irene M C van Amsterdam Marcellus Ubbink Oliver Einsle Albrecht Messerschmidt Angelo Merli Davide Cavazzini Gian Luigi Rossi Gerard W Canters 《Nature structural biology》2002,9(1):48-52
The transfer of electrons between proteins is an essential step in biological energy production. Two protein redox partners are often artificially crosslinked to investigate the poorly understood mechanism by which they interact. To better understand the effect of crosslinking on electron transfer rates, we have constructed dimers of azurin by crosslinking the monomers. The measured electron exchange rates, combined with crystal structures of the dimers, demonstrate that the length of the linker can have a dramatic effect on the structure of the dimer and the electron transfer rate. The presence of ordered water molecules in the protein-protein interface may considerably influence the electronic coupling between redox centers. 相似文献
10.
11.
The Sco family of proteins are involved in the assembly of the dinuclear CuA site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-Å resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function. 相似文献
12.
Barrett ML Harvey I Sundararajan M Surendran R Hall JF Ellis MJ Hough MA Strange RW Hillier IH Hasnain SS 《Biochemistry》2006,45(9):2927-2939
Rusticyanin from the extremophile Thiobacillus ferrooxidans is a blue copper protein with unusually high redox potential and acid stability. We present the crystal structures of native rusticyanin and of its Cu site mutant His143Met at 1.27 and 1.10 A, respectively. The very high resolution of these structures allows a direct comparison with EXAFS data and with quantum chemical models of the oxidized and reduced forms of the proteins, based upon both isolated and embedded clusters and density functional theory (DFT) methods. We further predict the structure of the Cu(II) form of the His143Met mutant which has been experimentally inaccessible due to its very high redox potential. We also present metrical EXAFS data and quantum chemical calculations for the oxidized and reduced states of the Met148Gln mutant, this protein having the lowest redox potential of all currently characterized mutants of rusticyanin. These data offer new insights into the structural factors which affect the redox potential in this important class of proteins. Calculations successfully predict the structure and the order of redox potentials for the three proteins. The calculated redox potential of H143M ( approximately 400 mV greater than native rusticyanin) is consistent with the failure of readily available chemical oxidants to restore a Cu(II) species of this mutant. The structural and energetic effects of mutating the equatorial cysteine to serine, yet to be studied experimentally, are predicted to be considerable by our calculations. 相似文献
13.
Leys D Meyer TE Tsapin AS Nealson KH Cusanovich MA Van Beeumen JJ 《The Journal of biological chemistry》2002,277(38):35703-35711
The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners. 相似文献
14.
M Lundeen 《Journal of inorganic biochemistry》1986,27(3):151-162
Statistical preferences of the side chains in proteins for the helical conformation, the beta strand conformation, and the turn conformation were derived from the computer-assigned secondary structures of 55 protein chains reported by Levitt and Greer in 1977 after averaging redundant structures and weighting structures that showed greater than 25% homology by w = square root N/N. Separate preferences are reported for free cysteine side chains and for cystine. These preferences are used to predict the secondary structures of cucumber plastocyanin and cupredoxin, whose crystal structures will be reported soon. The loops in the C-terminal copper-binding regions are discussed. 相似文献
15.
K Mann W Sch?fer U Thoenes A Messerschmidt Z Mehrabian R Nalbandyan 《FEBS letters》1992,314(3):220-223
We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30-37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence. 相似文献
16.
A. M. Nersissian C. Immoos M. G. Hill P. J. Hart G. Williams R. G. Herrmann J. S. Valentine 《Protein science : a publication of the Protein Society》1998,7(9):1915-1929
The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation. 相似文献
17.
The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans. 相似文献
18.
Byung-Cheon Jeong Si Hoon Park Kyoung Shin Yoo Jeong Sheop Shin Hyun Kyu Song 《Biochemical and biophysical research communications》2013,430(1):265-271
The single cystathionine β-synthase (CBS) pair proteins from Arabidopsis thaliana have been identified as being a redox regulator of the thioredoxin (Trx) system. CBSX1 and CBSX2, which are two of the six Arabidopsis cystathione β-synthase domain-containing proteins that contain only a single CBS pair, have close sequence similarity. Recently, the crystal structure of CBSX2 was determined and a significant portion of the internal region was disordered. In this study, crystal structures of full-length CBSX1 and the internal loop deleted (Δloop) form are reported at resolutions of 2.4 and 2.2 Å, respectively. The structures of CBSX1 show that they form anti-parallel dimers along their central twofold axis and have a unique ~155° bend along the side. This is different from the angle of CBSX2, which is suggestive of the flexible nature of the relative angle between the monomers. The biochemical data that were obtained using the deletion as well as point mutants of CBSX1 confirmed the importance of AMP-ligand binding in terms of enhancing Trx activity. 相似文献
19.
Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV. 相似文献