Evaluation of ¹³C and ¹H Fermi contact shifts in horse cytochrome c The origin of the anti‐Curie effect

Abstract

Many ferricytochromes c exhibit a peculiar effect in which the 'H chemical shifts of the haem methyl groups appear in pairs and, although the paramagnetic shifts of the two groups with the larger shifts decrease with temperature, those of the pair with the smaller shifts actually increase. Recent NMR studies [Santos, H. and Turner, D. L. (1992) Eur. J. Biochem. 206, 721 -7281 gave ' H and I3C assignments for most of the haem substituents and the axial ligands in horse cytochrome c at 30°C and 50°C in both oxidation states. These data are used together with an empirically determined magnetic susceptibility tensor to evaluate the F e d contact contribution to the paramagnetic shift and hence map the delocalisation of the unpaired electron. The anti-Curie effect is explained by a Boltzmann distribution between partially filled porphyrin 3e(n) molecular orbitals with an energy difference of 3 W/mol. The fact that the energy gap is small with respect to the energy of binding to the electron transfer partners calls into question the significance of the asymmetry of the electron distribution in the electron transfer process.The 'H-NMR spectrum of horse ferricytochrome c takes on a dramatic appearance because two of the haem methyl resonances are shifted by about 30 ppm to low field while the other two are shifted only by about 5 ppm and barely emerge from the main envelope of signals. The large shifts have been assigned to methyls 3 and 8, situated in diametrically opposed pyrroles, and are assumed to be generated largely by the Fermi contact interaction [l]. This implies a strong asymmetry in the distribution of the delocalised unpaired electron which had been the subject of speculation concerning the pathway for electron transfer [2J. Fermi contact shifts are expected to show an inverse temperature dependence, and methyls 3 and 8 exhibit such Curie law behaviour approximately, but methyls 1 and 5 have paramagnetic shifts which actually increase with temperature. Such anticurie behaviour has been observed for many c-type ferricytochromes, including those from horse [3], Pseudomonas aeruginosa [41, Rhodospirillum rubrum [5], Rhodopseudomonas gelatinosa [6], Paracoccus denitrijicans [7], and Methylophilus methylotrophus cytochrome cL which is illustrated in Fig.l. The effect has become accepted as a characteristic for identifying haem methyl resonances close to the main envelope of signals, and yet it seems never to have been explained.Anti-Curie behaviour has also been observed in ferredoxins, in which the effect is caused by thermal population of excited electronic spin states [8]. A similar effect has been observed in horse ferricytochrome c at elevated temperatures when the native low-spin protein is in fast chemical exchange with partially denatured high-spin forms [9]. This work, however, concerns only the region of linear temperature dependence of chemical shifts in which protein remains in a pure low-spin (S = '/z) state.

Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c″ by 1H‐NMR

“…The small chemical-shift range found for the haem resonances (the most shifted haem resonance appears at about 18 ppm and the chemical-shift span of the methyl group resonances is only 9 ppm), is indicative of a relatively high symmetry for the electron-spin distribution. This is supported by the fact that the 2-methyl group and the 12-methyl group show only very slight anti-Curie effects [32]. Furthermore, both the axial histidines experience reasonably large hyperfine shifts, suggesting roughly similar interactions with the haem iron, in contrast to ferricytochrome b5, the only other bis-histidinyl haem protein for which the haem electronic structure has so far been published [33].…”

1H NMR Investigation of the Distal Hydrogen Bonding Network and Ligand Tilt in the Cyanomet Complex of Oxygen-avidAscaris suum Hemoglobin

“…The temperature dependence of the heme methyl shifts reveals that the 1-CH 3 , 5-CH 3 exhibit positive slopes that are steeper than Curie (T Ϫ1 ) behavior, whereas the 3-CH 3 , 5-CH 3 exhibit slopes that are negative or exhibits antiCurie behavior. This effect is expected on the basis of thermal population of the excited orbital state, where the lone spin on the iron becomes delocalized into pyrroles B and D (60,(67)(68)(69). Lastly, the magnetic axes reported above allow the determination of ␦ dip for the axial His, which, in turn, provides ␦ con for each of the positions, as shown in Table I.…”

Use of Paramagnetic NMR Probes for Structural Analysis in Cytochrome c3 from Desulfovibrio Vulgaris

“…In recent studies of ferricytochroine c3 from DvH, the "C resonances of the haem substituents were also identified . These assignments formed the basis for the elaboration of a theoretical model which describes the Fermi contact shifts of those carbon nuclei in terms of a molecular orbital of eg symmetry with a rhombic perturbation (Turner, 1993(Turner, , 1995Turner et al, 1995). Moreover, the study has shown that the Fermi contact shifts are directly related to the orientations of both axial ligands and hence that the axial ligands dominate the rhombic perturbation.…”