Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds

The heme group of all mammalian peroxidases is covalently linked to the pro tein matrix via two esterbonds, as we have recently shown by Fourier transf orm infrared (FTIR) difference spectroscopy [Kooter, I,M., Pierik, A.J., Me rkx, M., Averill, B.A,, Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. A m. Chem. Sec. 119, 11542-11543]. We have examined the effects of mutation o f Asp94 and Glu242, responsible for those eater bonds in myeloperoxidase, o n the spectroscopic properties and catalytic activity of this enzyme. Mutat ion of Asp94 in myeloperoxidase results in two species. The first species h as spectroscopic characteristics similar to that of wild-type myeloperoxida se. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of t he ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mu tant still has about 30% residual peroxidase activity while for the Asp94-- >Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with i ts neighbouring residue Met243, according to resonance Raman spectra, is re sponsible for the low symmetry of the heme group. Mutation of either of the se residues results in loss of the bowed distortion from the planar conform ation, and in a heme group with higher symmetry. For the Glu242-->Gln mutan t 8% residual peroxidase activity is found.