Arginine 177 is involved in Mn(II) binding by manganese peroxidase

Site-directed mutations R177A and R177K in the gene encoding manganese pero xidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. Th e mutant enzymes were expressed in P. chrysosporium during primary metaboli c 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 a nd resonance Raman spectra of the ferric stare were similar to those of the wild-type enzyme, indicating that the heme environment was not significant ly affected by the mutations at Arg177. Apparent K-m values for Mn-II were similar to 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K-m values for the substrates, H2O2 and ferrocya nide, and the k(cat) values for Mn-II and ferrocyanide oxidation were simil ar to those of the wild-type enzyme. The second-order rate constants for co mpound I (MnPI) reduction of the mutant MnPs by Mn-II were similar to 10-fo ld lower than for wild-type MnP. In addition, the K-D values calculated fro m the first-order plots of MnP compound II (MnPII) reduction by Mn-II for t he mutant enzymes were similar to 22-fold greater than for wild-type MnP. I n contrast, the first-order rate constants for MnPII reduction by Mn-II wer e similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnP I reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide we re not significantly changed. These results indicate that both the R177A an d R177K mutations specifically affect the binding of Mn, whereas the rate o f electron transfer from Mn-II to the oxidized heme apparently is not affec ted.