Role of arginine 177 in the Mn-II binding site of manganese peroxidase - Studies with R177D, R177E, R177N, and R177Q mutants

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 c hrysosporium by examining two mutants: R177A and R177K. We now report on ad ditional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purifie d to homogeneity. The molecular mass and the UV/ visible spectra of the fer ric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and pre servation of the heme environment. However, steady-state and transient-stat e 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 Arg 177 decrease binding of Mn-II to the enzyme. These lowered binding efficien cies, as observed with the R177A and R177K mutants, suggest that the salt-b ridge between Arg177 and the Mn-II binding ligand Glu35 is disrupted in the se new mutants. Decreased k(cat) values for Mn-II oxidation, decreased seco nd-order rate constants for compound I reduction (k(2app)), and decreased f irst-order rate constants for compound II reduction (k(3)) indicate that th ese new mutations also decrease the electron-transfer rate. This decrease i n rate constants for compounds I and II reduction was not observed in our p revious study on the R177A and R177K mutations. The lower rate constants su ggest that, even with high Mn-II concentrations, the Mn-II binding geometri es 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 indica te a role for Arg177 in promoting efficient Mn-II binding and oxidation by MnP.