Role of arginine 177 in the Mn II binding site of manganese peroxidase

Abstract

Previously, we reported that Arg177 is involved in Mn II binding at the Mn II binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild‐type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady‐state and transient‐state kinetic analyses demonstrate significantly altered characteristics of Mn II oxidation by these new mutant enzymes. Increased dissociation constants ( K d ) and apparent K m values for Mn II suggest that these mutations at Arg177 decrease binding of Mn II to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt‐bridge between Arg177 and the Mn II binding ligand Glu35 is disrupted in these new mutants. Decreased k cat values for Mn II oxidation, decreased second‐order rate constants for compound I reduction ( k 2app ), and decreased first‐order rate constants for compound II reduction ( k 3 ) indicate that these new mutations also decrease the electron‐transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high Mn II concentrations, the Mn II binding geometries may be altered in the Mn II binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient Mn II binding and oxidation by MnP.