Characterization and peroxidase activity of a myoglobin mutant containing a distal arginine.
The spectroscopic, conformational, and reactivity characteristics of the T67R variant of sperm whale myoglobin have been studied to assess the effects of introducing an arginine residue into the distal side of this protein, as occurs in the active site of heme peroxidases. The overall circular dichroism (CD) and NMR spectroscopic properties of various derivatives of the protein are little affected by the mutation. The mutant contains a high-spin ferric ion with a water molecule as the sixth ligand, which exhibits slightly enhanced acidity (pK(a)=8.43+/-0.03) with respect to the corresponding derivative of wild-type myoglobin (pK(a)=8.60+/-0.04). The presence of the distal arginine increases the affinity of the Fe(III) center for azide (K=(6.0+/-0.5)x10(4) M(-1)) and decreases that for imidazole (K=12.0+/-0.2 M(-1)), with respect to the wild-type protein (K=(5.0+/-0.1)x10(4) and 24.7+/-0.7 M(-1), respectively). The peroxidase activity of T67R and wild-type myoglobins has been studied with a group of phenolic substrates related to tyrosine. The mutant exhibits an increased rate of reaction with hydrogen peroxide (k=1550+/-10 versus 760+/-10 M(-1) x s(-1)) and a generally increased peroxidase activity with respect to wild-type myoglobin. Relaxation measurements of proton nuclei of the phenolic substrates in the presence of either the T67R variant or the wild-type protein show that binding of these molecules occurs at distances of 8-10 A from the iron center, that is, close to the heme pocket, except for p-cresol, which can approach the heme more closely and, therefore, probably enter into the distal cavity.
Redaelli
Cristina
C
Dipartimento di Chimica Generale, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy.
Monzani
Enrico
E
Santagostini
Laura
L
Casella
Luigi
L
Sanangelantoni
Anna Maria
AM
Pierattelli
Roberta
R
Banci
Lucia
L
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002942:Circular Dichroism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010544:Peroxidases / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014907:Whales
D009211:Myoglobin; D010636:Phenols; D001120:Arginine; D006861:Hydrogen Peroxide; D010544:Peroxidases