Structure of the azurin mutant Phe114Ala from<i>Pseudomonas aeruginosa</i>at 2.6 Å resolution

Lc, Tsai; Sjölin, L.; Langer,; Pascher, T.; Nar, Herbert

doi:10.1107/s0907444994005627

Cited by 28 publications

(26 citation statements)

References 23 publications

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“…[42][43][44] The experimental DDG value for N47L was obtained from the experimental data on Alcaligenes denitrificans azurin. [45] The calculated values were obtained by using the forward change (l = 0 to 1) from the reduced to the oxidised state (&) and the backward change (l = 1 to 0) from the oxidised to the reduced state (&). For the N47DH variant, only the forward calculation was performed (DDG = 12 kJ mol À1 ).…”

Section: à4mentioning

confidence: 99%

Calculation of the Redox Potential of the Protein Azurin and Some Mutants

et al. 2005

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Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values were used to compute relative redox potentials. The precision of the free-energy calculations with the lambda coupling-parameter approach is evaluated as function of the number and sequence of lambda values, the sampling time and initial conditions. It is found that the precision is critically dependent on the relaxation of hydrogen-bonding networks when changing the atomic-charge distribution due to a change of redox state or pH value. The errors in the free energies range from 1 to 10 k(B)T, depending on the type of process. Only qualitative estimates of the change in redox potential by protein mutation can be obtained.

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Section: à4mentioning

confidence: 99%

Calculation of the Redox Potential of the Protein Azurin and Some Mutants

et al. 2005

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“…This is not valid for the PA azurin mutant F114A [29]. The mutation was performed on residue Fl14, which exhibits a n-electron overlap with the copper ligand His117, to investigate its suggested role in the electron self-exchange reaction.…”

Section: Geometries Of Cupredoxin Copper Sitesmentioning

confidence: 99%

“…Copper sites that resuked from the replacement of the axial methionine by lysine, glutamic acid and histidine were found to have spectroscopic properties between those of type 1 and type 2 sites [46][47][48]. These sites were named type 1.5 sites [29,42], but a clear understanding of their properties has still been lacking.…”

Section: Geometries Of Cupredoxin Copper Sitesmentioning

confidence: 99%

Metal sites in small blue copper proteins, blue copper oxidases and vanadium-containing enzymes

Messerschmidt

1998

Structure and Bonding

102

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The coordination geometries of metal sites in cupredoxins, mutants and metal derivatives of cupredoxins, multi-copper oxidases and a vanadium-containing chloroperoxidase as derived from X-ray crystallography are described. Correlations with their spectroscopic, electrochemical, electron transfer and catalytic properties are discussed. X-ray crystallography, EPR and Resonance Raman spectroscopy of copper sites in cupredoxins and mutants have led to a classification ranging from type 1 trigonal, type 1 distorted tetrahedral, type 1.5 to type 2. The mutation of copper ligands in azurin or amino acids close to the copper site changes the redox potential in a range of_+140 mV, only. The high redox potential of rusticyanin of 680 mV (azurin, 380 mV) should be mainly due to the special protein environment of the copper site (high proportion of hydrophobic residues).The type 1 and trinuclear copper centres of the multi-copper oxidases ascorbate oxidase, laccase and ceruloplasmin are presented. The metal sites of type 2 depleted, fully-reduced, peroxide and azide forms of ascorbate oxidase, as determined by X-ray crystallography, are discussed in terms of the mechanistic properties of these enzymes.The first X-ray structure of a vanadium-containing protein, namely of a chloroperoxidase from the fungus Curvularia inaequalis, is briefly discussed. The protein fold is mainly a-helical with two four-helix bundles. In the X-ray structure, which is an azide:enzyme complex, the vanadium exhibits a simple unexpected coordination geometry, namely, a trigonal bipyramidal coordination with three non-protein oxygen ligands (VO3 group), one nitrogen ligand from a histidine and one nitrogen from the exogenous azide ligand.Keywords: protein crystallography, small blue copper proteins, multi-copper oxidases, vanadium-containing enzyme, electron transfer 1 plastocyanin from Populus nigra [33,34] plastocyanin from Enteromorpha prolifera [35] plastocyanin from Populus nigra, Hg substituted [36] pseudoazurin from Alcaligenesfaecalis [37,38] amicyanin from Paracoccus denitrificans [39] amicyanin from Thiobacillus versutus [40] cucumber basic protein [41] cucumber stellacyanin [6] azurin from AD containing cadmium [42] copper-containing nitrite reductase from Achromobacter cycloclastes [15] ascorbate oxidase from Cucurbita pepo medullosa [43] mercury substituted type 1 site

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“…These include, among others, a pathway through the so-called hydrophobic patch at the surface ligand His117 (Nar et al, 1991a;Tsai et al, 1995). In the metal site, the Ni atom has shifted slightly from the regular copper position to obtain a more pronounced tetrahedral coordination site.…”

Section: Discussionmentioning

confidence: 99%

“…The structure-function relationships for electron transfer in blue-copper proteins have therefore attracted many investigators during recent years. Kinetic measurements, NMR studies, fluorescence anisotropy decays and crystallographic investigations have been used to explore the electron entrance site on the protein surface, as well as the electron exit site and plausible pathways (Lappin, Segai, Weatherburn & Sykes, 1979;Petrich, Longworth & Fleming, 1986;Tsai et al, 1995). Electron-transfer theory for the longrange electron transfer in proteins involving electron tunneling through a polypeptide environment, has also been explored previously (Marcus & Sutin, 1985;Plato, Michel-Beyerle, Bixon & Jortner, 1989;Beratan, Betts & Onuchic, 1991;Farid, Moser & Dutton, 1993).…”

Section: Introductionmentioning

confidence: 99%

Structure of the azurin mutant nickel–Trp48Met fromPseudomonas aeruginosaat 2.2 Å resolution

Tsai¹,

Sjölin²,

Langer³

et al. 1995

Acta Cryst D

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The structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa has been determined by difference Fourier synthesis using phases from the wild-type azurin model. The final crystallographic R value is 0.170 for 17 394 reflections to a resolution of 2.2 A,. The mutant crystallized in the orthorhombic space group P212121, a = 57.4, b = 80.4, c = 110.3 A,. The four molecules in the asymmetric unit are packed as a dimer of dimers. The nickel metal site of this mutant structure is similar to the zinc metal site in the azurin Asp47 mutant. The site-specific mutation was performed at residue Trp48, which is located in the center of the protein in a highly hydrophobic environment, to investigate its suggested role in the long-range electrontransfer pathway between the disulfide bond on one side of the protein to the Cu centre. The structure around the mutation site Met48 showed no significant change compared with the wild-type structure.

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Structure of the azurin mutant Phe114Ala fromPseudomonas aeruginosaat 2.6 Å resolution

Cited by 28 publications

References 23 publications

Calculation of the Redox Potential of the Protein Azurin and Some Mutants

Calculation of the Redox Potential of the Protein Azurin and Some Mutants

Metal sites in small blue copper proteins, blue copper oxidases and vanadium-containing enzymes

Structure of the azurin mutant nickel–Trp48Met fromPseudomonas aeruginosaat 2.2 Å resolution

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