Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5·5 and pH 9·0

Nar, Herbert; Messerschmidt, Albrecht; Huber, Robert; Kamp, Mart van de; Canters, Gerard W.

doi:10.1016/0022-2836(91)80173-r

Cited by 565 publications

(703 citation statements)

References 33 publications

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“…Blue copper proteins show a so-called "alkaline transition" of their absorption wavelength, absorption intensity, and redox potential [91][92][93][94]. With increasing pH, especially at pH 8-11, the absorption maximum at ca.…”

Section: Properties Of Type I Copper In Multicopper Oxidasesmentioning

confidence: 99%

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Structure and function of type I copper in multicopper oxidases

Sakurai

Kataoka

2007

Cell. Mol. Life Sci.

128

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Abstract. The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordinating 1Met or the non-coordinating 1Phe/1Leu, and it exhibits spectral properties and an alkaline transition similar to those of the blue copper center in blue copper proteins. Since the type I copper center in multicopper oxidases is deeply buried inside the protein molecule, electron transfers to and from type I copper are performed through specific pathways: the hydrogen bond between an amino acid located at the substrate binding site and a His residue coordinating type I copper, and the His-Cys-His sequence connecting the type I copper center and the trinuclear copper center comprised of a type II copper and a pair of type III coppers. The intramolecular electron transfer rates can be tuned by mutating the fourth ligand of type I copper. Further, mutation at the Cys ligand gives a vacant type I copper center and traps the reaction intermediate during the four-electron reduction of dioxygen.

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Section: Properties Of Type I Copper In Multicopper Oxidasesmentioning

confidence: 99%

“…3 Å [17][18][19][20][21][22]. Figure 2 compares the structure and properties of the type I copper center and the blue copper center together with their variants, the red copper center with the coordination of 1Cys2His1Glu and a water molecule [5], and the coupled Cu A center in N 2 OR and COX [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Structure and function of type I copper in multicopper oxidases

Sakurai

Kataoka

2007

Cell. Mol. Life Sci.

128

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“…pseudoazurin Both the structures of pseudoazurin from A. faeealis S-6 and azurin from P. aeruginosa have been determined by Xray crystallographic analysis [4][5][6]. Although identity in amino acid sequence between the two blue-copper proteins is very low (11%), the X-ray analyses revealed that these two proteins…”

Section: Comparison Of the X-ray Structures Of Azurin Andmentioning

confidence: 99%

“…Xray analysis of pseudoazurin has revealed an 8-stranded [~-barrel structure followed by two m-helices at the C-terminus, binding a type 1 copper atom on the top of the [3-barrel [4,5]. The structure is surprisingly similar to that of another cupredoxin, azurin [6], although no significant identity in amino acid sequences of the two proteins was observed [7]. By using site-directed mutagenesis of pseudoazurin, we have previously shown that a 'ring' of lysine residues (Lys-10, Lys-38, Lys-57, and Lys-77) located at the edge of a hydrophobic region surrounding a type 1 copper site is involved in interaction with NIR [8].…”

Section: Introductionmentioning

confidence: 97%

Site‐directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron‐transfer complex with a copper‐containing nitrite reductase from Alcaligenes faecalis S‐6

et al. 1996

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Kinetic analysis of electron transfer between azurinfrom Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between coppercontaining proteins. Apparent values of kc,t and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoaznrin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, DI1K, P36K, and Dill(/ P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of DllK, P36K, and DllK/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of DllK and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

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“…At this point, the availability of detailed structural information from a closely analogous protein is of crucial help since it will be used to discriminate which protons are likely to be dipolarly connected with previously assigned protons. We have used the structures of the Ni(1I) and Zn(I1) azurins [34,351, as well as the wild-type Cu(I1) protein at low and high pH [29] during the assignment process. The analysis of NOESY, COSY and TOCSY correlations between protons from the second coordination sphere is performed in a more conventional way [54] and permits finding and specifically assigning spin systems which can be, in turn, dipolarly connected with previously assigned hyperfine-shifted signals.…”

Section: Methodsmentioning

confidence: 99%

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

Salgado

Jiménez

Donaire

et al. 1995

European Journal of Biochemistry

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Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 'H-NMR spectrum of Pseudornonas aeruginosa Co(I1)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(I1)-azurin, i.e. His46, Hisll7, Cysll2, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(I1) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N,S plane towards the Gly45 0 axial position and weakly interacting with the Met121 sulfur.Keywords. Azurin; paramagnetic NMR; metal substitution; type 1 copper proteins ; cobalt proteins.Substitution of the naturally occumng metals in metalloproteins is used as a common strategy in inorganic biochemistry [l, 21. In a first approach it can be considered as a kind of sitedirected mutation, the most simple we can think of in the case of a metalloprotein. In this sense, apart from the possible function of the metal, it can give an idea of the metal site selectivity and structural rigidity, the mechanism of metal uptake, some general chemical properties of the metalloprotein, etc. In some special cases metal substitution is used to probe the metal site [ 1 -31. This is possible when the chromophore that brings along the exogenous metal has peculiar spectroscopic properties. For the blue copper proteins, like azurin, the natural copper(I1) center has itself singular, informative and in some way exciting spectroscopic properties (41. These are principally EPR and electronic spectral characteristics whose extensive study throughout the last few decades has achieved a detailed knowledge of type 1 copper sites [4-61. But even in this case metal substitution has proved to be useful in solving fundamental questions such as the assignment of the main visible bands of the blue copper proteins as S(Cys)-metal charge transfer bands Abbreviations. COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy ; WEFT, water-eliminated Fo...

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Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5·5 and pH 9·0

Cited by 565 publications

References 33 publications

Structure and function of type I copper in multicopper oxidases

Structure and function of type I copper in multicopper oxidases

Site‐directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron‐transfer complex with a copper‐containing nitrite reductase from Alcaligenes faecalis S‐6

¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

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Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5·5 and pH 9·0

Cited by 565 publications

References 33 publications

Structure and function of type I copper in multicopper oxidases

Structure and function of type I copper in multicopper oxidases

Site‐directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron‐transfer complex with a copper‐containing nitrite reductase from Alcaligenes faecalis S‐6

1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

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¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin