Reactivity of manganese peroxidase: site-directed mutagenesis of residues in proximity to the porphyrin ring.

The purpose of this study was to determine the effect of heme pocket hydrophobicity on the reactivity of manganese peroxidase. Residues within 5 A of the heme active site were identified. From this group, Leu169 and Ser172 were selected and mutated to Phe and Ala, respectively. The mutant proteins were then characterized by steady-state kinetics. Whereas the Leu169Phe mutation had little, if any, effect on activity, the Ser172Ala mutation decreased kcat and also the specificity constant (kcat/Km) for Mn2+, but not H2O2. Transient-state studies indicated that the mutation affected only the reactions of compound II. These results indicate that compound II is the most sensitive to changes in the heme environment.