¹H-NMR Study of Temperature-Induced Structure Alteration at the Active Site of Horse Heart Cytochrome c

Abstract

The molecular structure of the active site of horse heart met-cyano cytochrome c, as a function of temperature, has been investigated using ¹H-NMR. A temperature dependence study of the NMR spectra revealed that one heme methyl proton resonance exhibits anti-Curie behavior, i.e., the hyperfine shift increases with increasing temperature. Analyses of the average heme methyl proton hyperfine shift and the proximal His imidazole proton resonances indicated that the iron-His bonding interaction in this protein is essentially independent of temperature. Since such an anomalous temperature dependence of the heme methyl proton resonance disappears in met-cyano complex of a heme peptide prepared by enzymatic degradation of the protein [Smith, M. and McLendon, G. (1981) J. Am. Chem. Soc. 103,4912-4921], the anti-Curie behavior observed for the heme methyl proton resonance in met-cyano cytochrome c is attributed to a rotational displacement of the heme about the iron-His bond relative to the protein moiety due to a temperature-dependent conformational alteration of the heme-protein linkage. Such rotational mobility of heme at the active site of a protein may be responsible for the anomalous temperature dependence of heme methyl proton hyperfine shifts reported for many c-type ferri cytochromes.

Curie plot, cytochrome c, hemoprotein, hyperfine shift, NMR