Proton NMR investigation of the heme active site structure of an engineered cytochrome c peroxidase that mimics manganese peroxidase.
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 been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and (15)N shift of the bound C(15)N(-) 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 of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely.
Department of Chemistry, University of Illinois at Urbana-Champaign 61801, USA.
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D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003486:Cyanides / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007930:Leucine / Q000737:chemistry; D018716:Molecular Mimicry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010544:Peroxidases / Q000737:chemistry*; D015202:Protein Engineering; D011522:Protons; D013053:Spectrophotometry; D014633:Valine / Q000235:genetics
D003486:Cyanides; D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D006639:Histidine; D001120:Arginine; D010544:Peroxidases; C051129:manganese peroxidase; D003578:Cytochrome-c Peroxidase; D007930:Leucine; D014633:Valine; D005998:Glycine