title: Role of axial ligands in the reactivity of Mn peroxidase from Phanerochaete chrysosporium.
abstract: Site-directed mutagenesis was performed on Mn peroxidase (MnP) from the white-rot fungus Phanerochaete chrysosporium to investigate the role of the axial ligand hydrogen-bonding network on heme reactivity. D242 is hydrogen bonded to the proximal His of MnP; in other peroxidases, this conserved Asp, in turn, is hydrogen bonded to a Trp. In MnP and other fungal peroxidases, the Trp is replaced by a Phe (F190). Both residues are thought to have a direct influence on the electronic environment of the catalytic center. To study only the active mutants at D242 and F190, we used degenerate oligonucleotides allowing us to screen all 19 possible amino acid mutants at these positions. Two mutants at D242 passed our screen, D242E and D242S. Both mutations impaired only the functioning of compound II. The reactions of the ferric enzyme with H(2)O(2) were unaffected by the mutations, as were the reactions of compound I with reducing substrates. The D242S and D242E mutations reduced the first-order rate constant for the reaction of MnP compound II with chelated Mn(2+) from 233 s(-1) (wild type) to 154 s(-1) and 107 s(-1), respectively. Three F190 mutants passed our screen, F190V, F190L, and F190W. Similar to mutants at D242, these mutants largely affected the function of compound II. The F190V mutation increased the first-order rate constant for the reduction of compound II by chelated Mn(2+) to 320 s(-1). The F190L mutation decreased this rate to 137 s(-1). The F190W mutant was not very stable, but at pH 6.0, this mutation decreased the rate of compound II reduction by Mn(2+) from 140 s(-1) in the wild type to 36 s(-1). There was no indication that the F190W mutant was capable of forming a protein-centered Trp cation radical. All the mutations altered the midpoint potential of the Fe(3+)/Fe(2+) couple of the enzyme, as calculated from cyclic voltammagrams of the proteins. The values were shifted from -96 mV in the wild-type enzyme to -123 mV in D242S, -162 mV in D242E, -82 mV in F190L, -173 mV in F190V, and -51 mV in F190W. Collectively, these results demonstrate that D242 and F190 in MnP influence the electronic environment around the heme and that the reactions of compound II are far more sensitive to this influence than the reduction of compound I.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Whitwam
authors.forename: R S
authors.initials: RS
authors.lastname: Koduri
authors.forename: M
authors.initials: M
authors.lastname: Natan
authors.forename: M
authors.initials: M
authors.lastname: Tien
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D004563:Electrochemistry; D004795:Enzyme Stability / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D020075:Phanerochaete / Q000201:enzymology*; D010649:Phenylalanine / Q000235:genetics; D013053:Spectrophotometry; D014364:Tryptophan / Q000235:genetics
chemical_list: D008024:Ligands; D001224:Aspartic Acid; D010649:Phenylalanine; D014364:Tryptophan; D010544:Peroxidases; C051129:manganese peroxidase