Glu375Gln and Asp225Val mutants: About the nature of the covalent linkagesbetween heme group and apo-protein in bovine lactoperoxidase

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C,, Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lac toperoxidase the effect of mutation of Asp225 and Glu375, the residues thou ght to be responsible for the covalent binding of the heme group to the apo protein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K., Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in co rrespondence of those residues, the mutants Asp225Val and Glu375Gln were ex pressed in CHO cells and their products purified and characterized. Unequiv ocal evidence about the existence of ester linkages as well as their relati ve contribution to the specific spectroscopic and catalytic properties of b LPO is here discussed. (C) 2001 Elsevier Science Ltd. All rights reserved.