'''MANGANESE BINDING MUTANT IN CYTOCHROME C PEROXIDASE'''
===MANGANESE BINDING MUTANT IN CYTOCHROME C PEROXIDASE===
==Overview==
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The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.
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==About this Structure==
==About this Structure==
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[[Category: Peroxidase]]
[[Category: Peroxidase]]
[[Category: Protein engineering]]
[[Category: Protein engineering]]
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Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase., Wilcox SK, Putnam CD, Sastry M, Blankenship J, Chazin WJ, McRee DE, Goodin DB, Biochemistry. 1998 Dec 1;37(48):16853-62. PMID:9836578
