Analysis of the Temperature Dependence of the 1 H and 13 C Isotropic Shifts of Horse Heart Ferricytochrome c: Explanation of Curie and Anti-Curie Temperature Dependence and Nonlinear Pseudocontact Shifts in a Common Two-Level Framework Lucia Banci, ² Ivano Bertini,* ,² Claudio Luchinat, ‡ Roberta Pierattelli, ² Nikolai V. Shokhirev, §,| and F. Ann Walker* ,§ Contribution from the Department of Chemistry, UniVersity of Florence, Via Gino Capponi 7, 50121 Florence, Italy, the Department of Soil Science and Plant Nutrition, UniVersity of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy, and the Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721 ReceiVed January 23, 1998 Abstract: The 1 H and 13 C hyperfine shifts of the heme methyls of horse heart ferricytochrome c have been measured over the temperature range 278-328 K in order to interpret the “anomalous” temperature-dependence of the hyperfine shifts in terms of their pseudocontact and contact shifts. By taking advantage of the available pseudocontact shifts for protein nuclei measured at 303 and 323 K (Santos, H.; Turner, D. L. Eur. J. Biochem. 1992, 206, 721-728), the metal-centered pseudocontact shifts have been analyzed in terms of a thermally accessible excited state lying 355-590 cm -1 to higher energy which has a magnetic susceptibility tensor with the rhombic anisotropy, Δ rh , which is rotated by 90° to that of the ground state. The metal-centered pseudocontact shifts have been evaluated at all temperatures at which the chemical shifts were measured, and these calculated values were used to evaluate the contact shifts of each heme methyl for the two nuclei. The temperature dependence of the heme methyl contact shifts for both 1 H and 13 C, assuming a thermally accessible excited state, was then used to evaluate the spin density for the four -pyrrole heme carbons to which the methyls are attached. The ligand-centered pseudocontact shifts have been estimated and found to give a modest contribution to the experimental behavior. The 1 H and 13 C data are highly self-consistent. The present analysis provides deep insight into the electron distribution on the porphyrin ring in low-spin Fe(III) hemes. Introduction Since Kowalsky’s pioneering report of the 56.4-MHz proton NMR spectrum of horse heart cytochrome c in 1962 1 there have been many extensive studies of various aspects of the NMR spectra of a number of cytochromes c. 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Soc. 1998, 120, 8472-8479 S0002-7863(98)00261-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/05/1998