Indexed on: 28 Jul '99Published on: 28 Jul '99Published in: Biochemistry
The mutants at position 242 of manganese peroxidase (MnP), where the native Asp has been substituted with a Ser or a Glu, have been shown to be active, and are here characterized by electronic, EPR, and NMR spectroscopies. We have also mutated another residue on the proximal side, Phe 190 to Val and Leu, yielding active mutants. When studied by the above-mentioned spectroscopies, the mutants at both positions 242 and 190 exhibit three pH-dependent transitions. In contrast to the transitions observed at low and high pH, the spectroscopic studies reveal that the transition at intermediate pH has pK(a) values up to 2 units lower for the mutants at D242E and -S and F190V than for the wild type. This process is due to the ionization of a group that affects the transition to the bis-histidine coordination at the iron. The observed changes in the pK(a) values are related to the altered affinity of the calcium-binding site in the distal pocket. Other variations are observed in the other two pK(a) values. Characterization of the cyanide derivatives indicates that the location and orientation of the distal and proximal His residues are essentially identical to that in the wild type. Our results indicate that mutations on the proximal side residues can affect changes in the distal side. In particular, deprotonation of a group, whose pK(a) is influenced by the nature of the residues in the proximal side, produces a movement of helix B, which in turn induces the coordination of the distal His and the loss of the distal calcium ion.