Structural characterization of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants

Murphy, Loretta M.; Strange, R.W.; Karlsson, Bengt; Lundberg, Lennart; Pascher, Torbjörn; Reinhammar, Bengt; Hasnain, S.S.

doi:10.1021/bi00059a013

Cited by 61 publications

(73 citation statements)

References 38 publications

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“…A closer approach of Gly45 C = O to the Cu ion than in wt Cu(I1) azurin has been observed, for instance, in the crystal structure of Zn azurin . A strengthening of the axial Cu-0 = C Gly45 interaction, which is weak in wt azurin (Nar et al, 1991b;Lowery and Solomon, 1992), is compatible with the observed changes of the All and gll values and of the wavelengths of the CT bands in the EPR and the optical spectra, respectively, of M44K azurin at high pH (Peisach and Blumberg, 1974;Karlsson et al, 1991;Pascher et al, 1993;Murphy et al, 1993;Romero et al, 1993). The observed shift of the 281-cm-' peak in the M44K-azurin RR spectrum by +4 cm-' (Fig.…”

Section: Spectroscopic Effects Of Lys44 Deprotonationmentioning

confidence: 53%

Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue‐copper protein azurin from Pseudomonas aeruginosa

Kamp

Canters

Andrew

et al. 1993

European Journal of Biochemistry

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The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-+Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation.Electrostatic interactions have an important role in the determination of both the structure and function of proteins (Warshel and Russel, 1984;Davis and McCammon, 1990;Bashford, 1991). In the case of redox proteins, electrostatic interactions have a marked influence, e.g. on the midpoint potential (Rees, 1985;Moore et al., 1986). In this connection, the engineering of charged residues can be of particular interest, as it may lead to distinctly interpretable effects (Sternberg et al., 1987;Cutler et al., 1989;Varadarajan et al., 1989;Rodgers and Sligar, 1991). In this study, the effects of the replacement of the uncharged residue Met44 of the blue-copper protein azurin from Pseudomonas aeruginosa by a lysine residue (Van de Kamp et al., 1990a, b) on the electron self-exchange (ESE) rate constant (kESE), the spectroscopic and electrochemical properties, as well as their pH dependence, are reported (for reviews on blue-copper proteins see Adman, 1991 ;Sykes, 1991 ;Solomon et al., 1992).Met44 is located in the so-called hydrophobic surface patch of azurin (Nar et al., 1991 a, b; Fig. 1). This patch covers the type-] Cu site of azurin, the Cu ion being located at approximately 0.7 nm below the surface. The Cu-ligand resi- due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) MATERIALS AND METHODSIsolation and purification of azurin P. aeruginosa wt azurin was isolated from Escherichia coli as described (Van de Kamp et al., 1990~). The M44K mutant of azurin was isolated from E. cot...

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Section: Spectroscopic Effects Of Lys44 Deprotonationmentioning

confidence: 53%

Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue‐copper protein azurin from Pseudomonas aeruginosa

Kamp

Canters

Andrew

et al. 1993

European Journal of Biochemistry

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“…X-ray absorption spectra at the Cu and Zn K edges were measured in¯uorescence, again on SRS Station 9.2. Data-collection procedures have been described previously (Murphy et al, 1993). The EXAFS was normalized to a unit metal atom and extracted from the background absorption using the Daresbury Laboratory program EXBACK (Morrell et al, 1989).…”

Section: X-ray Fluorescence and Exafs Data Collectionmentioning

confidence: 99%

X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 Å resolution

Ellis

Dodd

Strange

et al. 2001

Acta Crystallogr D Biol Crystallogr

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Copper‐containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer–monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8. The structure of this nitrite reductase has previously been determined at pH 4.6. It has now been crystallized under new conditions at pH 8.5. Its crystallographic structure provides a structural explanation for the greatly reduced activity of the enzyme at high pH. Characterization of overexpressed protein in solution by EXAFS suggested that the protein lacked Cu in the catalytic type 2 Cu site and that the site was most probably occupied by Zn. Using the anomalous signals from Cu and Zn, the crystal structure revealed that the expressed protein was devoid of Cu in the catalytic site and that only a trace amount (<10%) of Zn was present at this site in the crystal. Despite the close structural similarity of the catalytic site to a number of Zn enzymes, these data suggest that Zn, if it binds at the catalytic copper site, binds weakly in nitrite reductase.

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“…Since the copper ion is common, the structures of the copper binding sites must determine the chemical differences between the cupredoxins. Many structures have been determined for mutant (Nar et al, 1991b;Murphy et al, Cupredoxins include rusticyanin (RC), azurin from Alcaligenes denitrificans (AZAd ), cucumber basic protein (CBP), Plastocyanin from Populus nigra (PCPn ), pseudoazurin (cAZ), and amicyanin (AC). Secondary structure assignments were made using the algorithm of Kabsch and Sander (1983) as implemented in RIBBONS (Carson & Bugg, 1986) using the available three-dimensional coordinates for cupredoxins as defined in the legend to Figure 2.…”

Section: The Copper Binding Sitementioning

confidence: 99%

Multiple Wavelength Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly Oxidizing Cupredoxin with Extreme Acid Stability

Walter

Ealick

Friedman

et al. 1996

Journal of Molecular Biology

124

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Structural characterization of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants

Cited by 61 publications

References 38 publications

Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue‐copper protein azurin from Pseudomonas aeruginosa

Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue‐copper protein azurin from Pseudomonas aeruginosa

X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 Å resolution

Multiple Wavelength Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly Oxidizing Cupredoxin with Extreme Acid Stability

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