Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221-->Met mutant.
A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221-->Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pK(a) of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by (1)H-NMR data, and the loss of the pi-pi interaction between His-170 and Phe-221.
Howes
B D
BD
Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, 50121 Firenze, Italy.
Veitch
N C
NC
Smith
A T
AT
White
C G
CG
Smulevich
G
G
D017360:Arabidopsis; D002384:Catalysis; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids; D013059:Spectrum Analysis, Raman
D006877:Hydroxamic Acids; D008024:Ligands; D006418:Heme; D010649:Phenylalanine; D008715:Methionine; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid