Reduction potentials and their pH dependence in site‐directed‐mutant forms of azurin from Pseudomonas aeruginosa

Abstract

A spectroelectrochemical method has been used to determine the reduction potential of the copper site in wild-type and 22 mutant forms of azurin from Pseudomonas aeruginosa at 25 "C and in the range pH 4-8; the effect of buffers and ionic strength on the potentials has also been studied. Amino-acid residues changed include Metl21, which provides an S atom at a distance of about 0.3 nm from the metal, some amino acids in the hydrophobic patch, other residues believed to be important in electron transfer with physiological partners and some internal amino acids. The observed potentials span a range of about 300 mV. In all cases the potentials increase with decreasing pH, but the pK, values describing the pH dependence are essentially unchanged except in three mutants, where they change by pH 0.6-1.1 (up in one and down in two). The largest potential changes were found in some Met121 mutants, at which position large hydrophobic residues raise the potential, whereas negatively charged residues lower it; a decreased potential is also found in the Met121 + End mutant, which probably has H,O coordinated to the metal. Gly45 has its carbonyl group coordinated to copper, but the potential of Gly45 3 Ala is close to that of the wild type. Some substitutions in the hydrophobic patch cause an increase in the potential, whereas substitutions involving His35 and Glu91 do not result in significant changes. No single mechanism for tuning the potential of the copper site can be discerned, but in many cases there are probably indirect effects of the protein conformation causing changes in metal-ligand interactions.Azurin belongs to a family of small blue-copper proteins which function in electron-transfer chains in plants and bacteria (Adman, 1985). These proteins have unique spectral properties, such as intense absorbance bands around 600 nm and a narrow hypefine splitting in the CuZ+ EPR signal, and they also have unusually high reduction potentials among Cu2+ complexes. A perplexing fact is, however, that these potentials can vary by more than 0.5 V between members of the family despite the fact that the spectroscopic properties are relatively constant. Since the intense blue color and specific EPR signal are properties of the oxidized proteins, it has been suggested that proteins with a high reduction potential have a preferential stabilization of the Cu+ form (Gray and Malmstrom, 1983). To test this hypothesis, or other possible mechanisms for tuning the potentials, is one of the purposes of the program in site-directed mutagenesis of which the study described here is part.We have prepared mutant forms of azurin with substitutions of a metal ligand (Metl21) (Karlsson et al., 1991), of surface residues, some believed important in the interactions with electron-transfer partners , and also of some residues localized more internally. In this study we report the determination by thin-layer spectroelectrochemisCorrespondence to T. Vanngird,

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

“…It also supports the conclusion above that a water molecule is not embedded in the cavity. A polarizable water molecule close to the copper ion would lower the potential, as in the mutant where a stop codon was introduced in position 121 (Pascher et al, 1993). This leads to an open structure allowing water entry which results in a reduction potential reduced by over one third (from 310 to 205 mV).…”

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

“…Since the optical spectra of all the mutated azurins were principally similar, although small differences in the spectral parameters were observed, we concluded that the environment around type 1 copper is not greatly altered by the mutations. Differences of + 3 nm in maximum absorption are commonly seen for azurin mutants of surface residues [21]. Redox potentials of wild-type and mutated azurins were determined by redox titration in 0.1 M potassium phosphate buffer at pH 7.0.…”

Engineering type 1 copper sites in proteins

“…Replacement of Met"' in the azurins tunes the EO over a range of -105 mV to +138 mV with respect to the wt value at pH 7.0 [21,69]. Replacing His1i7 or His46 in the azurins by a glycine and supplying the Cu site with external ligands can increase the midpoint potential by about 100 mV [57].…”

“…Replacements in the second coordination sphere of the Cu can have fairly drastic affects on E,,, the causes of which are not clearly understood. For instance, the Pro*'Ala mutation in pseudoazurin causes a change of + 139 mV of E. [68], while the Asn47Leu and Asn47Asp mutations in azurin cause changes by + 110 and +23 mV, respectively [67,69]. Study of the temperature dependence of the Asn47Leu mutant vs. the wt, moreover has shown that the observed AE, between wt and mutant can strongly vary with temperature due to a sizeable entropy contribution to the difference in E, [67].…”