Redesign of Cytochrome c Peroxidase into a Manganese Peroxidase:  Role of Tryptophans in Peroxidase Activity

Abstract

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I'. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1. k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0. 599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.

Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: Nature and possible function of compound ES☆

“…Similar results were also presented from recent EPR studies of the reactions of PAA with WT KatG and the variants, Y249F and R439A/N [30]. Analogous to our results with P450cam variants, the porphyrin p-cation radical of Cpd I of cytochrome c peroxidase (CcP) is reduced more slowly to Cpd ES with the double mutant W51F/W191F than with WT [31,32]. Using a series of sequential mutations of the two tryptophans, Trp51 and Trp191, and the six tyrosines, Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236 in CcP, it was shown that the rate constant for Cpd I converting to Cpd ES decreases from 57 s À1 to 7 s À1 [31].…”

Spectroscopic evidence for an engineered, catalytically active Trp radical that creates the unique reactivity of lignin peroxidase

“…The same mechanism of a diffusible mediator has been proposed for the oxidation of lignin by MnP, except that in this enzyme, a loosely bound Mn 2ϩ ion having a binding site close to the heme site is the species reacting with lignin, once oxidized to Mn 3ϩ by MnP. Interestingly, an engineering strategy similar to the one described in this work was applied to design MnP activity into a cytochrome c peroxidase (CcP) protein scaffold (6)(7)(8).…”

Lignocellulose biotechnology: issues of bioconversion and enzyme production

“…A number of designer enzymes, also called glycosynthases, including cellulases and hemicellulases have been engineered by replacing the nucleophilic residue thus resulting in higher yields of different oligosaccharides (Palcic, 1999;Fairweather et al, 2002). Substituting two tryptophan residues in the heme cavity by two phenylalanines in a cytochrome-c peroxidase resulted in improved activity (Gengenbach, 1999). Increasing the thermostability of recombinant manganese peroxidase has been attempted by engineering a disulfide bridge contiguous to the distal Ca +2 binding site (Reading and Aust, 2000).…”