X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin.
The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)
Max-Planck Institut für Biochemie, Abteilung Strukurforschung, Martinsried bei München, Germany.
A
A
Romero
C W
CW
Hoitink
H
H
Nar
R
R
Huber
A
A
Messerschmidt
G W
GW
Canters
D000421:Alcaligenes / Q000235:genetics; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D008667:Metalloproteins / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D014961:X-Ray Diffraction
D008667:Metalloproteins; D010940:Plant Proteins; D001400:Azurin; C003343:stellacyanin protein, plant; D003300:Copper; D008715:Methionine