13C NMR spectroscopy of core heme carbons as a simple tool to elucidate the coordination state of ferric high-spin heme proteins

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Evidence is presented demonstrating that the magnitudes of the 13C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13C shifts centered at approximately -80 ppm. The relatively small spectral window (400 to -100 ppm) covering the meso-13C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295-302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774-6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13C NMR spectroscopic approach is demonstrated using hexa- and pentacoordinate myoglobin. Subsequently, 13C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate. © 2006 American Chemical Society.

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Inorganic Chemistry

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