The role of Glu39 in Mn-II binding and oxidation by manganese peroxidase from Phanerochaete chrysoporium

Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-ro t fungi and is part of the extracellular lignin degrading system in these o rganisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn-II and efficiently release Mn-III. Initial site-directed mutage nesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-typ e Mn binding and rate of oxidation, calling into question the role of E39 a s an Mn ligand. To investigate this hypothesis, we performed computer model ing studies which indicated metal-ligand bond distances in the E39D variant and in an E35D-E39D-D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D Variants u sed in the previous study, as well as an E39A single variant and the E35D-E 39D-D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn-II binding (K -m increases 20-30-fold) and Mn-II oxidation (k(cat) decreases 50-400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The cata lytic efficiency of the E39A variants decreased similar to 10(4)-fold, whil e that of the E39D variant decreased similar to 10(3)-fold. Contrary to ini tial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of t he triple variant decreased only 10(4)-fold, which is similar to 10(2)-fold better than that reported for the E35Q-D179N double variant. These combine d studies indicate that precise geometry of the Mn ligands within the Mn bi nding site of MnP is essential for the efficient binding, oxidation, and re lease of Mn by this enzyme. The results clearly indicate that E39 is a Mn L igand and that mutation of this ligand decreases both Mn binding and the ra te of Mn oxidation.