The Met99Gln Mutant of Amicyanin fromParacoccusversutus

Abstract

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic 1H NMR study of Co(II)-pseudoazurin

“…All the hyperfine shifted resonances follow a Curie temperature dependence, as observed in other Co(II)-substituted blue copper proteins [15,24,25,26,31,32,33,34,35,36,37]. Their chemical shifts and relaxation rates are typical of signals corresponding to proton nuclei from metal ligands ( Table 1).…”

“…Resonances A and C are broader than the rest of the signals in the spectrum, and exhibit shorter T 1 values (Table 1). Based on these spectral features, we feel confident to assign A and C to the Cys78 b-CH 2 protons by analogy with similar assignments in other Co(II)-substituted cupredoxins [15,24,25,26,31,32,33,34,35,36,37].…”

“…Paramagnetic NMR investigations on cupredoxins include studies of the native Cu(II) species [19,20,21,22,23] and of their Co(II) and Ni(II) derivatives [15,24,25,26,27,28,29,30,31,32,33,34,35,36,37]. These studies have afforded evidence on the interplay between the strength of the metal-axial ligand interaction and the metal-Cys covalence [15,20,21].…”

Effects of axial ligand mutation of the type I copper site in bilirubin oxidase on direct electron transfer-type bioelectrocatalytic reduction of dioxygen

“…Site-directed point mutation of the axial ligand has been carried out for blue copper proteins. It has been reported that the replacement of Met at the axial ligand by Gln causes the negative shift in the formal potential of the type I Cu site in blue copper proteins [7][8][9][10][11]. In contrast, the replacement of Gln by Met causes the positive shift in the formal potential [12,13].…”

The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

“…Azurin has been the topic of many such studies [5]. Amicyanin from P. versutus has also been studied and the effect of mutation of its axial Met ligand to Gln was examined [6]. In those studies the metal specificity and role of the metal during protein folding was studied by first denaturing the protein usually with guanidine HCl.…”