A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K-m values for the substrate Mn-II that were similar to 50-fold greater than the corresponding K-m value for the wild-type enzyme. Likewise, the k(cat) values for Mn-II oxidation were similar to 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K-m value for Mn-II was similar to 120-fold greater, and the k(cat) value was similar to 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by Mn-II allowed the determination of the equilibrium dissociation constants (K-D) and first-order rate constants for the mutant proteins. The K-D values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were similar to 200-fold and similar to 4000-fold less, respectively, than that of the wild-type enzyme. In constrast, the K-m values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.