Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.
In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies <nu(Cu-S)>, calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.
Durão
Paulo
P
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2781-901 Oeiras, Portugal.
Chen
Zhenjia
Z
Silva
Catarina S
CS
Soares
Cláudio M
CM
Pereira
Manuela M
MM
Todorovic
Smilja
S
Hildebrandt
Peter
P
Bento
Isabel
I
Lindley
Peter F
PF
Martins
Lígia O
LO
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D007532:Isoleucine / Q000378:metabolism; D042845:Laccase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D007532:Isoleucine; D003300:Copper; D042845:Laccase; D007930:Leucine