Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome
cperoxidase (C
cP) containing a Mn(II)-binding site reported previously (MnC
cP; see Yeung, B. K.-S., etal. (1997)
Chem. Biol. 5, 215-221). The goal of the present study is to elucidate the role of tryptophansin peroxidase activity since C
cP contains both Trp51 and Trp191 while manganese peroxidase (MnP)contains phenylalanine residues at the corresponding positions. The presence of Trp191 in C
cP allowsformation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical calledcompound I'. The absence of a tryptophan residue at this position in MnP is the main reason for theformation of an intermediate called compound I which contains a ferryl oxo and porphyrin
![](/images/gifchars/pi.gif)
-cation radical.In this study, we showed that introduction of the Trp191Phe mutation to MnC
cP did not improve MnPactivity (specific activity: MnC
cP, 0.750
![](/images/entities/mgr.gif)
mol min
-1 mg
-1; MnC
cP(W191F), 0.560
![](/images/entities/mgr.gif)
mol min
-1 mg
-1.
kcat/
Km: MnC
cP, 0.0517 s
-1 mM
-1; MnC
cP(W191F), 0.0568 s
-1 mM
-1) despite the fact that introductionof the same mutation to WTC
cP 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 livedcompound I in WTC
cP (decay rate, 18 s
-1), but also significantly improved MnP activity in MnC
cP(MnC
cP(W51F, W191F): specific activity, 8.0
![](/images/entities/mgr.gif)
mol min
-1 mg
-1;
kcat/
Km, 0.599 s
-1 mM
-1). The increasein activity can be attributed to the Trp51Phe mutation since MnC
cP(W51F) showed significantly increasedMnP activity relative to MnC
cP (specific activity, 3.2
![](/images/entities/mgr.gif)
mol min
-1 mg
-1;
kcat/
Km, 0.325 s
-1 mM
-1). Aswith MnP, the activity of MnC
cP(W51F, W191F) was found to increase with decreasing pH. Our resultsdemonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizingcompound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity becausethis mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determiningstep in the reaction mechanism.