EPR of azurins from pseudomonas aeruginosa and alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in pseudomonas aeruginosa protein

Groeneveld, C. M.; Aasa, Roland; Reinhammar, Bengt; Canters, Gerard W.

doi:10.1016/0162-0134(87)80059-4

Cited by 35 publications

(28 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In native blue copper proteins, the intensity of this band is usually low [38,39]. The EPR spectra show an increase in hyperfine splitting ( A z ) from 0.0062 cm −1 in wild‐type A. denitrificans azurin [40] to 0.0102 cm −1 in Met121His azurin [22], suggesting a lower delocalization of the unpaired electron [36]. This may reduce the rate of electron transfer via Cys112, although the point corresponding to Met121His in Fig.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Role of ligand substitution on long‐range electron transfer in azurins

Farver

Jeuken

Canters

et al. 2000

European Journal of Biochemistry

View full text Add to dashboard Cite

Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR 2 radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin±imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways.Keywords: azurin; electron transfer; enthalpy±entropy compensation; Marcus theory; pulse radiolysis.Electron transfer plays an important role in many biological systems, and a central question is to what extent do specific structural properties of proteins affect the rates of electron transfer [1±6]. The blue single-copper protein, azurin, which serves as an electron mediator in certain bacteria, has become an effective model for the study of intramolecular long-range electron transfer (LRET) in proteins [7±12]. The 3D structures have been determined for a large number of wild-type and single-site-mutated azurins [13±18] and shown to consist of a rigid b-sheeted polypeptide. As it contains two potential redox centres, i.e. the copper ion co-ordinated directly to amino acid residues and a disulfide bridge (RSSR) at opposite ends of the barrel-shaped molecule, no additional external redox group is required to study internal LRET. Indeed, we have previously demonstrated that LRET between these two centres can be induced by pulse-radiolytic single-electron reduction of RSSR [7±12].We have examined the effect of specific structural changes on the rate of intramolecular electron transfer between the RSSR 2 radical and the Cu(II) centre in both wild-type and single-site-mutated azurins. In the present study, we used a series of azurin mutants in which modifications were introduced into the copper ligand sphere. Two mutants (His46Gly and His117Gly) contain a free co-ordination site. In the absence of external ligands, water will bind at the copper site [19]. Alternatively, in the presence of excess imidazole, the latter may take up the vacant position at th...

show abstract

Section: Discussionmentioning

confidence: 99%

“…This should be compared with the corresponding coupling in wild‐type Ps. aeruginosa azurin (0.0058 cm −1 ) [40]. As for Met121His azurin, the higher value of the hyperfine splitting suggests a lower delocalization of the unpaired electron.…”

Section: Discussionmentioning

confidence: 99%

Role of ligand substitution on long‐range electron transfer in azurins

Farver

Jeuken

Canters

et al. 2000

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The variations with pH are known to be caused by the (de)protonation of His35 [16]. They are compatible with tiny changes in the copper-site geometry equivalent to variations of 1" or less in the angles between Cu-ligand bonds [21]. The small difference between wt azurin and [M64E]azurin is eliminated when the pH is lowered to 3 (below the pK, of Glu64) and the charge on the carboxyl group of Glu64 is neutralized by protonation.…”

Section: Discussionmentioning

confidence: 99%

The introduction of a negative charge into the hydrophobic patch of Pseudomonas aeruginosa azurin affects the electron self‐exchange rate and the electrochemistry

Pouderoyen

Mazumdar

Hunt

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

By changing Met64 into a glutamate by means of site-directed mutagenesis a negative charge was introduced into the hydrophobic patch of azurin from Pseudomonas aeruginosa. The three-dimensional structure of the protein and the structure of the metal site in particular, appear unaffected by the mutation. The observed change of the midpoint potential of the mutant of 28 mV is ascribed to the deprotonation of Glu64. The electron-self-exchange rate constant equals that of the wild-type protein at pH 4.5 but decreases by almost two orders of magnitude at high pH. Electron transfer is inhibited only when both of the reacting azurin molecules have an ionized glutamate at position 64 in their hydrophobic patch. Electron transfer at a graphite electrode is slowed down by the presence of the negative charge in the hydrophobic patch. The results demonstrate once again that the Culigand His117 in the hydrophobic patch is the likely entry and exit point for electrons. This observation holds both for the (homogeneous) electron-self-exchange reaction in solution as well as for the (heterogenous) reaction at an electrode.The mechanism of electron transport inside redox proteins and enzymes is the subject of strong debate at the moment (for a recent review, see [I]). Do specific and welldefined electron-transfer pathways exist within a protein, or is electron transfer a random event from a structural point of view and does the protein merely act as an organic glass [2] it is noted that the electron-transfer efficiency is probably enhanced by a water molecule that in the crystal structure appears immobilized with respect to the protein through H-bonds to His117 and Val43 [lo]. This water molecule is likely to be retained in the solution structure [ll, 121. However, there was an ambiguity in the interpretation of the results from the site-directed mutagenesis study in the sense that not only the electron-transfer efficiency of the [M44K]-azurin but also the spectroscopic and structural properties of the Cu-site were affected by the mutation. The observed slowing down of the electron-transfer rate might, therefore, have to be ascribed to the change of the Cu-site instead of the modification of the hydrophobic patch. As Met44 is located very close to the Cu-site (Met44 is adjacent to Hisll7, see Fig. 1) it was thus decided to study a second mutant in which another residue in the hydrophobic patch, Met64, more remote from His117 ( Fig. 1) was mutated. Instead of a lysine, a negatively charged residue, Glu64, was introduced. The mutant was characterized, its structure was studied, and the electron-transfer properties were investigated as a function of pH. The results are reported here. MATERIALS AND METHODS Bacterial strains and plasmidsEscherichia coli strain JMlOl [I41 was used for cloning and expression of the Pseudomonas aeruginosa azu gene.

show abstract

“…This property has been related to the protonation/deprotonation of a specific histidine residue (His35), that is not a copper ligand, but belongs to the second coordination sphere of the metal. The deprotonation of this residue causes a considerable reorientation of its sidechain [18] and a subsequent reorganization of the threedimensional structure of the protein, with changes in the second coordination sphere of the copper, as determined by nuclear magnetic resonance (NMR), extended X-ray absorption fine structure (EXAFS) and EPR studies [1,[19][20][21]. However, in Alcaligenes (Al.)…”

Section: Inorganic Biochemistrymentioning

confidence: 99%