Arginine 177 Is Involved in Mn(II) Binding by Manganese Peroxidase

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Abstract
Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV−vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent Km values for MnII were ∼20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent Km values for the substrates, H2O2 and ferrocyanide, and the kcat values for MnII and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by MnII were ∼10-fold lower than for wild-type MnP. In addition, the KD values calculated from the first-order plots of MnP compound II (MnPII) reduction by MnII for the mutant enzymes were ∼22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by MnII were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from MnII to the oxidized heme apparently is not affected.