Evidence is presented demonstrating that the magnitudes of the C-13 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 C-13 meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit C-13 shifts centered at approximately -80 ppm. The relatively small spectral window ( 400 to - 100 ppm) covering the meso-C-13 shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare C-13-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 C-13 NMR spectroscopic approach is demonstrated using hexa- and pentacoordinate myoglobin. Subsequently, C-13 NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae ( ShuT) is pentacoordinate.