Site-directed mutations at phenylalanine-190 of manganese peroxidase: effects on stability, function, and coordination.
A series of site-directed mutants, F190Y, F190L, F190I, and F190A, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium was generated by overlap extension with 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 manganese peroxidase variants (MnPs) were purified and characterized by kinetic and spectroscopic methods. At pH 4.5, the UV-vis spectra of the ferric and oxidized states of the mutant proteins were very similar to those of the wild-type enzyme. Steady-state kinetic analyses showed that the apparent Km and k(cat) values for MnII and H2O2 also were similar to the corresponding values for the wild-type MnP. The apparent Km and k(cat) values for ferrocyanide oxidation by MnP were not affected by the F190Y, F190L, or F190I mutations; however, the apparent Km value for ferrocyanide oxidation by the F190A mutant MnP was approximately 1/8 of that for the wild-type enzyme. Likewise, the apparent k(cat) value for ferrocyanide oxidation by the MnP F190A mutant was approximately 4-fold greater than the corresponding k(cat) for the wild-type MnP. The stabilities of both the native and oxidized states of MnP were significantly affected by several of the mutations at Phe190. Replacement of Phe190 by either Ile or Ala significantly destabilized the resultant proteins to thermal denaturation. Moreover, the rates of spontaneous reduction of the oxidized intermediates, MnP compounds I and II, were dramatically increased for the F190A mutant relative to the rates observed for the wild-type enzyme. The spectroscopic properties of the wild-type and F190 mutant MnPs were examined as a function of pH. At room temperature, increasing pH from 5.0 to 8.5 induced a FeIII high- to low-spin transition for all of the MnP proteins. This transition may involve direct coordination of the distal His residue to the heme iron to produce bishistidinyl coordination as suggested by magnetic circular dichroism spectroscopy. The pH at which this transition occurred was considerably lower for the F190A and F190I variants and suggests that Phe190 plays a critical role in stabilizing the heme environment of MnP.
Kishi
K
K
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Hildebrand
D P
DP
Kusters-van Someren
M
M
Gettemy
J
J
Mauk
A G
AG
Gold
M H
MH
D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005295:Ferrocyanides / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D013053:Spectrophotometry; D013696:Temperature
D005295:Ferrocyanides; D007527:Isoenzymes; D006418:Heme; D010649:Phenylalanine; D010544:Peroxidases; C051129:manganese peroxidase; C020354:hexacyanoferrate II