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 horseradis h 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-t ype enzymes can be detected spectroscopically, These include a change in th e Fe(III) resting state of the enzyme to an unusual quantum mechanically mi xed-spin haem species, a marked decrease in the pK(a) of the alkaline trans ition and a reduction in enzyme stability at alkaline pH for both Fe(III) a nd Fe(II) forms. The perturbation of the haem pocket in the mutant can be a ttributed to several factors, including the increased steric freedom and so lvent accessibility of the His-170 ligand, as indicated by H-1-NMR data, an d the loss of the pi-pi interaction between His-170 and Phe-221.