RESCUE OF HIS-42-]ALA HORSERADISH-PEROXIDASE BY A PHE-41-]HIS MUTATION - ENGINEERING OF A SURROGATE CATALYTIC HISTIDINE

Formation of the ferryl (Fe-IV=O) porphyrin radical cation known as Co mpound 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 mutatio n was shown earlier to decrease the rate of Compound I formation by a factor of similar to 10(6) and of guaiacol oxidation by a factor of si milar to 10(4), In contrast, an F41A mutation has little effect on per oxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1 995) J. Biol. Chem. 270, 19430-19438). We report here construction, ex pression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maxim um at similar to pH 6.3. Addition of H2O2 produces a Compound I specie s (lambda(max) = 406 nm) that is reduced by 1 eq of K4Fe(CN)(6) to the ferric state (lambda(max) = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the proc ess, 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, Th e k(cat) values for guaiacol oxidation by wild-type, H42A, and F41H/H4 2A HRP are 300, 0.015, and 1.8 s(-1). The corresponding k(cat) values for ABTS oxidation are 4900, 0.41, and 100 s(-1), respectively. These results show that a histidine at position 41 substitutes, albeit imper fectly, for His-42 in peroxidative turnover of the enzyme, The F41H/H4 2A double mutant has peroxidative properties intermediate between thos e of the native enzyme and the H42A mutant. The F41H/H42A double mutan t, however, is a considerably better thioanisole sulfoxidation and sty rene epoxidation catalyst than native or H42A HRP, The surrogate catal ytic residue introduced by the F41H mutation thus partially compensate s for the H42A substitution used to increase access to the ferryl oxyg en.