| Abstract
| - The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung,B. K., et al. (1997) Chem. Biol. 4, 215−221] that closely mimics manganese peroxidase (MnP) has beencharacterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonancesfrom the heme pocket as well as resonances from catalytically relevant amino acid residues in the congesteddiamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadeningpattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center isfirmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in nodetectable structural changes on the distal heme pocket of the protein. However, notable structural changesare observed at the proximal side of the heme cavity. Both CεH shift of the proximal histidine and 15Nshift of the bound C15N- suggest a weaker heme Fe(III)−N(His) bond in MnCcP compared to WtCcP.Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)−N(His) bond strength ofthe template protein CcP so that its bond strength is similar to that of the target protein MnP. The resultspresented here help elucidate the impact of designing a metal-binding site on both the local and globalstructure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP thatmimics MnP more closely.
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