title: Rescue of His-42 --> Ala horseradish peroxidase by a Phe-41 --> His mutation. Engineering of a surrogate catalytic histidine.
abstract: Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of approximately 10(6) and of guaiacol oxidation by a factor of approximately 10(4). In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at approximately pH 6.3. Addition of H2O2 produces a Compound I species (lambdamax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (lambdamax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 x 10(4) M-1 s-1. This is to be compared with 0.9 x 10(7) M-1 s-1 for wild-type HRP and 19 M-1 s-1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s-1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s-1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
authors.forename: M I
authors.initials: MI
authors.lastname: Savenkova
authors.forename: S L
authors.initials: SL
authors.lastname: Newmyer
authors.forename: P R
authors.initials: PR
authors.lastname: Montellano
mesh_terms: D000409:Alanine / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D006639:Histidine / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
chemical_list: D011994:Recombinant Proteins; D010649:Phenylalanine; D006639:Histidine; D006899:Mixed Function Oxygenases; D006735:Horseradish Peroxidase; C022333:peroxygenase; D000409:Alanine