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Zoppellaro G Harbitz E Kaur R Ensign AA Bren KL Andersson KK 《Journal of the American Chemical Society》2008,130(46):15348-15360
Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other. 相似文献
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《Journal of Electroanalytical Chemistry》1987,217(1):129-140
The direct electrochemistry of the single heme cytochrome c551 from the bacterium Pseudomonas aeruginosa has been investigated at gold electrodes surface-modified through chemisorption of polyfunctional organic molecules. The results have been compared and contrasted with those obtained under the same conditions for the eukaryotic cytochrome c from horse heart. Both cytochromes give a quasi-reversible electrode reaction at pH 6.0 at a modified interface presenting only 4-pyridyl groups to the solution suggesting the occurrence, in both cases, of a hydrogen bonding interaction from lysine side-chains on the protein to pyridyl-nitrogens on the electrode surface. However, in contrast, gold electrodes modified by Pyridine-n-AldehydeThioSemicarbazones (n = 2, 3, 4) give electrochemistry which is strongly isomer-dependent in the case of horse heart cytochrome c but completely isomer-independent in the case of cytochrome c551. It is suggested that interaction of the eukaryotic protein with surfaces is dominated by its lysine residues only, but that interaction of the bacterial cytochrome is through hydrogen bonding from the surface to both lysines and carboxylate groups of aspartate residues. This is supported by observation of the loss of cytochrome c551 electrochemistry at 4-pyridyl-only modified gold at pH 9.0 compared with the good, quasi-reversible electrochemistry maintained under the same conditions at PATS-4 modified gold. It is concluded that, while the two cytochromes show many similarities with respect to their structures and functions, they have quite different interfacial electron transfer reactions, particularly at PATS-modified electrodes. This may correlate with the known large differences between the two proteins in net electrostatic charge and surface charge distribution. 相似文献
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Garner DK Vaughan MD Hwang HJ Savelieff MG Berry SM Honek JF Lu Y 《Journal of the American Chemical Society》2006,128(49):15608-15617
The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center. 相似文献
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Heme oxygenase (HO), an amphipathic microsomal protein, catalyzes the oxygen-dependent degradation of heme (iron-protoporphyrinIX) to alpha-biliverdin, CO, and free iron ion. Interestingly, all of HO regiospecifically oxidize the alpha-meso position of the heme to form alpha-biliverdin isomer while nonenzymatic heme degradation forms all four possible alpha-, beta-, gamma-, delta-biliverdin isomers at nearly identical yield. Recently, an interesting example has been found in HO (PigA) of the Gram-negative bacterium Pseudomonas aeruginosa, which does not produce alpha-biliverdin at all, but forms the mixture of beta- and gamma-biliverdins at a ratio of 3:7. While studying the mechanism of the unique regioselectivty of PigA, we found essential amino acid residues, Lys34, Lys132, and Phe189, controlling the unique regioselectivity of PigA. In this communication, we show that Lys34 and Lys132 are essential amino acid residues to hold the rotated heme in the active site of PigA via hydrogen-bonding interaction with the heme propionate and that Phe189 controls the product ratio of beta- and delta-biliverdins via steric interaction with heme substituents. These interactions place the beta- or delta-meso position of the heme at the oxidation site of PigA, leading to the unique regioselectivity. 相似文献
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Calculations on the structure and spectral properties of cytochrome c551 using DFT and ONIOM methods
The active site geometry of cytochrome (Cyt) c(551) and its mutated form containing Fe(II) and Fe(III) ions have been calculated using density functional theory (DFT)-based Becke's three-parameter hybrid exchange and Lee-Yang-Parr correlation (B3LYP) method. In addition, calculations have also been carried out using hybrid meta DFT-based M06 functional. The effect of the protein milieu on the active site geometry has also been probed using two-layer via our own N-layered integrated molecular orbital + molecular mechanics (ONIOM) method. Evidence from the calculations reveal that the active site geometry is not significantly affected by the oxidation state of metal ion. The difference in the geometry of the active site and that of the same with the entire protein environment is only minimal, which shows that the protein milieu does not influence the structure of the active site. The calculated electronic transition energies from the time-dependent DFT (TDDFT) calculations are in close agreement with the experimental values. Although there are no significant variations in the active site geometry upon oxidation, the changes in the electronic transition energies have been attributed to the reduction in the overlap of metal ion with the ligand orbitals. In addition, it is found that mutation does not influence the active site geometry and the electronic transition energies. Nevertheless, mutation leads to the formation of more compact structure than the native Cyt c(551). 相似文献
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M R?nnberg 《Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry》1976,30(8):721-726
Kinetic studies of the reaction mechanism of Pseudomonas cytochrome c peroxidase (PaCCP) were made by the method of product inhibition using oxidized cytochrome C (551 P.aeruginosa) and oxidized Pseudomonas azurin as products. Inhibition by the two oxidized substrates was linearly non-competitive towards the respective reduced electron donor and towards hydrogen peroxide. Although a full kinetic analysis is experimentally impossible in a peroxidase-type reaction, the results do provide some evidence in favour of an ordered reaction mechanism in which hydrogen peroxide is the first to add to PaCCP and electron donor the second. 相似文献
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Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp) Bound to Synthetic Metal Complexes
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Chikako Shirataki Dr. Osami Shoji Mitsuyoshi Terada Prof. Dr. Shin‐ichi Ozaki Dr. Hiroshi Sugimoto Prof. Dr. Yoshitsugu Shiro Prof. Dr. Yoshihito Watanabe 《Angewandte Chemie (International ed. in English)》2014,53(11):2862-2866
The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasAp) can capture several synthetic metal complexes other than heme. The crystal structures of HasAp harboring synthetic metal complexes revealed only small perturbation of the overall HasAp structure. An inhibitory effect upon heme acquisition by HasAp bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasAp bound to iron–phthalocyanine inhibits heme acquisition in the presence of heme‐bound HasAp as an iron source. 相似文献
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Casalini S Battistuzzi G Borsari M Bortolotti CA Ranieri A Sola M 《The journal of physical chemistry. B》2008,112(5):1555-1563
The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest. 相似文献
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R Soininen N Kalkkinen 《Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry》1977,31(7):604-608
The effect of pH on the oxidized Pseudomonas cytochrome c peroxidase molecule was studied by measuring the peroxidatic activity, the sedimentation velocity, the circular dichroic spectra in the far UV and Soret regions, and the optical absorption spectra of the enzyme in the pH range 2.5-13.0 at a constant ionic strength (micron = 0.1). The enzyme was stable in a narrow pH region, pH 6.0 - 7.4. In the low pH range the gross tertiary structure was observed to change quite simultaneously with the enzymatic activity and secondary structure. The optical absorption spectra indicated that there were no coordinated internal protein liqands in the 6th coordination positions of the heme prosthetic groups at the lowest pH studied. In the high pH range the secondary structure and the protein environment of hemes were observed to remain stable after the tertiary structure had changed and the activity had decreased. According to the optical absorption spectra the 6th internal protein ligands of hemes were retained at the highest pH studied. 相似文献
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M R?nnberg N Ellfolk 《Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry》1975,29(7):719-727
The steady state kinetics of cytochrome c peroxidase from Pseudomonas aeruginosa (PaCCP) has been studied by initial velocity techniques using several cytochromes c (550 and 555 P. aeruginosa; 551 P. fluorescens) and Pseudomonas azurin as electron donors and hydrogen peroxide as electron acceptor. From the initial velocity patterns a sequential mechanism with compulsory substrate-binding order is proposed for PaCCP. A comparative kinetic study of the peroxidatic oxidation of cytochrome c-551 (P. aeruginosa) by yeast cytochrome c peroxidase was made to evaluate the significance of electrostatic interactions in complex formation between the enzyme and substrates. 相似文献
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Molecular recognition between two redox partners, azurin and cytochrome c 551, is studied at the single-molecule level by means of atomic force spectroscopy, after optimizing azurin adsorption on gold via sulfhydryl-terminated alkanethiol spacers. Our experiments provide evidence of specific interaction between the two partners, thereby demonstrating that azurin preserves biorecognition capability when assembled on gold via these spacers. Additionally, the measured single-molecule kinetic reaction rate results are consistent with a likely transient nature of the complex. Interestingly, the immobilization strategy adopted here, which was previously demonstrated to favor electrical coupling between azurin (AZ) and the metal electrode, is also found to facilitate AZ interaction with the redox partner, if compared to the case of AZ directly adsorbed on bare gold. Our findings confirm the key role of a well-designed immobilization strategy, capable of optimizing both biorecognition capabilities and electrical coupling with the conductive substrate at the single-molecule level, as a starting point for advanced applications of redox proteins for ultrasensitive biosensing. 相似文献
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