Cassette mutagenesis of Met121 in azurin from <i>Pseudomonas aeruginosa</i>

Karlsson, Bengt; Nordling, Margareta; Pascher, Torbjörn; Tsai, Li-Chu; Sjölin, L.; Lundberg, Lennart

doi:10.1093/protein/4.3.343

Cited by 117 publications

(122 citation statements)

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“…Contributions from an azide-Cu charge-transfer band cannot be excluded, since such bands appear in this region with considerable intensity (103 M-Icm i). However, a similar spectrum is observed for the Lys121 mutant at pH 7 demonstrating that optical bands in this region do not require charged ligands (Karlsson et al, 1991).…”

Section: The Azjde Derivative Of the Metl21ala Mutantsupporting

confidence: 61%

“…This was supported by the fact that stellacyanin, the only type 1 blue protein known to lack a methionine in the apical direction at that time, has the lowest reduction potential (184 mV) of the bluecopper proteins (Fields, Guss & Freeman, 1991). The importance of methionine for tuning the redox potential has later been confirmed in a cassette mutagenesis study conducted on this residue in azurin from Pseudomonas aeruginosa (Karlsson et al, 1991). The Metl21 residue was mutated into all the other 19 amino-acid residues and the reduction potential of 13 of these mutants was measured.…”

Section: Introductionmentioning

confidence: 89%

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Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Tsai¹,

Bonander²,

Harata³

et al. 1996

Acta Cryst D

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The crystal structures of the azurin mutant Met l21Ala and its azide derivative Met l21Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met l21Ala mutant, the distance between the copper ion and His117 increases by 0.34/~ compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74/~. In the Met l 21Ala-azide structure, the azide anion occupies the cavity created by replacing the Met l21 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A, towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met l21Ala mutant still maintains the properties of an ordinary type 1 copper site while the Metl21Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

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Section: The Azjde Derivative Of the Metl21ala Mutantsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 89%

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Tsai¹,

Bonander²,

Harata³

et al. 1996

Acta Cryst D

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“…The location of the copper with respect to the plane in the case of the blue AxNiR is, therefore, of some interest. Its position significantly closer to the strong ligand plane and similar to that found in azufins may account for the differences in colour of two sub-classes of Cu-NiR' s. The green colour of the Glul21 mutant of azurin from P. aeruginosa (Karlsson et al, 1991;Karlsson, 1993;Strange, Murphy, Karlsson, Reinhammar & Hasnain, 1996) may also thus arise from a similar movement of the Cu from the ligand plane. In fact, the crystal structure of Glul21 mutant shows a displacement of Cu of ,-~0.4 A (Karlsson, 1993).…”

Section: Comparison Of Cu Sites In Axnir With Green Nir'smentioning

confidence: 80%

Structures of a Blue-Copper Nitrite Reductase and its Substrate-Bound Complex

Dodd¹,

Hasnain²,

Abraham³

et al. 1997

Acta Cryst D

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Copper-containing nitrite reductases (NiR's) have been conveniently subdivided into blue and green NiR's which are thought to be redox partners of azurins and pseudo-azurins, respectively. Crystal structures of two green NiR's have recently been determined. Alcaligenes xylosoxidans has been shown to have a blue-copper nitrite reductase (AxNiR) and two azurins with 67% homology both of which donate electrons to it effectively. The first crystal structure of a blue NiR (AxNiR) in its oxidized and nitrite-bound forms, with particular emphasis to the Cu sites, is presented. The Cu-Smet distance is the same as those in the green NiR's. Thus, the length of this interaction is unlikely to be responsible for differences in colour. Crystallographic data presented here taken together with structural data of other single Cu type-1 proteins and their mutants suggest that the displacement of Cu from the strong ligand plane is perhaps the cause for the differences in colour observed for otherwise 'classical' blue Cu centre. Nitrite is observed binding to the catalytic Cu in a bidentate fashion displacing the water molecule, offering a neat rationalization for the XAFS observation that the type-2 Cu-ligand distances increase on nitrite binding as a result of increased coordination. These results are discussed in terms of enzyme mechanism.

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“…Presumably the mutation has not significantly changed the electronic properties of the site. Examples are provided by mutants in which Met"' has been replaced by a non-polar residue (Ala, Val, Ile, Leu) [55] and by the mutants His117Gly and His46Gly, when the gap in the structure created by the mutation is filled by external ligands like substituted imidazoles or anions (see Fig. 2) [29,56,57].…”

Section: (A) 'Blue Type 1' Sitesmentioning

confidence: 99%

“…Examples of green type 1 sites are provided by mutants in which Met12' has been replaced by a ligand like His, Lys or Glu (pH 7) [55]. Also the His"7Gly mutant at pH 6 belongs to this category.…”

Section: (C) 'Green Type 1' Sitesmentioning

confidence: 99%

Engineering type 1 copper sites in proteins

1993

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The use of site-directed mutagenesis methods has revolutionalized the study of the so-called type 1 and type 2 copper sites in proteins. In particular our understanding of the relation between the structure, and the mechanistic and spectroscopic features of these sites is benefitting from the application of these techniques. Recent progress in the field is reviewed with emphasis on the study of type 1 sites. Topics covered comprise the characteristics of the natural type 1 and type 2 sites, the genetics of blue copper proteins, the modification of Cu sites, the spectroscopy of natural and engineered type 1 and type 2 sites, the effect of mutations on midpoint potentials and the mechanism of electron transfer as carried out by the blue copper proteins.

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Cassette mutagenesis of Met121 in azurin from Pseudomonas aeruginosa

Cited by 117 publications

References 0 publications

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Structures of a Blue-Copper Nitrite Reductase and its Substrate-Bound Complex

Engineering type 1 copper sites in proteins

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