Crystal structures of substrate binding site mutants of manganese peroxidase

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of Mn-II to Mn-III. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed Mn-II binding site of MnP consists of a heme propionate, three acidic Ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of Mn-II bound to the protein. Crystals grown in the absence of Mn-II, or in the presence of EDTA, exhibited diminished electron density at this site, Crystals prawn in excess Mn-II exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major Mn-II binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined, The mutant structures lack a cation at the Mn-II binding site. The structure of the Mn-II binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.