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

Abstract

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...

Native CuA redox sites are largely resilient to pH variations within a physiological range

“…Most likely the different pH dependencies of Tt-Cu A and Cu A -Azu can be partially ascribed to the protonable His35 residue in Cu A -Azu, which is very close to the active site. 21,22 Our results highlight the importance of the protein matrix acting as a second (and higher) coordination sphere in regulating both the electronic and redox properties of the Cu A redox centre, which is consistent with the differential role of the weak axial ligand methionine in regulating thermodynamic and kinetic ET parameters, as reported recently. 8,14 Moreover, they challenge the notion of redox properties of the primary electron acceptor of CcO being fine-tuned by local variations of pH in vivo.…”

Anti-cooperativity in the two electron oxidation of the S118C disulfide dimer of azurin

“…The bimolecular electron transfer rate of B 10 4 M − 1 s − 1 between the two molecules of S118C azurin dimer is also slow, as compared with wild type azurin ( 10 6 M − 1 s − 1 ). The hydrophobic patch surrounding His117 has been shown to be the site of interaction for intermolecular electron transfer [1][2][3][4]. As a result of the dimerization this patch is expected to be no longer exposed to the solvent and, thus, to have become inaccessible for other dimers to form association complexes.…”

“…Although the structure of the binary complex in the e.s.e. reaction is unknown, NMR studies have shown that the hydrophobic patch surrounding the surface exposed copper ligand His117 is the site of interaction [1][2][3][4]. A clear demonstration of how the association of two molecules along this patch may occur, is provided by the crystal structure, where two water molecules connect the two His117 copper ligands via hydrogen bonds [5,6].…”