¹³C and proton NMR studies of horse cytochrome c

Abstract

The CH, groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both I3C-NMR and 'H-NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side-chain methyl and the 21 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 3C resonances were obtained through shift-correlation experiments and comparison with earlier 'H-NMR studies, by further measurements of proton-proton interactions, or by elimination.Comparison of the paramagnetic shifts of carbon and protons indicates a small redox-related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30 -50 "C.The early demonstration of the ability to assign resonances of individual carbon nuclei which do not have attached protons in I3C-NMR spectra of proteins [l] led to a short period of intensive investigation. More recently, developments in two-dimensional NMR spectroscopy have stimulated new interest in I3C studies by offering a highly efficient alternative to selective proton decoupling experiments for correlating chemical shifts of 13C nuclei with the chemical shifts of directly attached protons [2]. The simultaneous determination of the I3C and 'H chemical shifts of individual groups provides much greater resolution than can be achieved in studies of a single nucleus and also allows the distinction of methyl, methylene and methine groups without resorting to isotopic enrichment [3, 41. We therefore set out to explore the utility of such methods for studying the natural abundance 13C spectra of proteins and the value of 13C-NMR data in seeking to define a possible redox-related change in the structure of horse cytochrome c.The characterisation of the methyl groups of horse cytochrome c was described in earlier letters which also discussed the possibility of obtaining primary assignments of resonances by measuring the temperature dependence of diamagnetic chemical shifts [4, 51. The present study employs the selective suppression of methylene resonances as a step towards characterising the remaining resonances of carbon nuclei with attached protons. Careful measurement of 13C chemical shifts and of their temperature dependence from onedimensional spectra is shown to be sufficient to determine primary assignments in many cases.The application to horse cytochrome c might be regarded simply as a model study, since a wealth of data has been obtained recently from 'H-NMR studies alone [6 -91. However, the assignment of the proton spectrum is not yet complete and the confirmation of assignments by independent methods Correspondence to D. L. Turner, Department of Chemistry, The University, Southampton SO9 5NH, UK.is generally desirable. This work has generated several additions and corrections to earlier proton assignments as well as the large number of new 13C assignments.The extensive assignment of I3C resonances in the NMR spectra of fer...

NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c

“…If this were the primary contribution to the NMR HFS of the heme meso 13 C resonances, then the sign would be positive 79. Instead, based on the average heme meso 13 C shift of reduced horse heart cyt c (97.4 ppm),80 the sign of the heme meso 13 C HFS is negative (Table 2). Importantly, negative spin density at the meso carbons of a low-spin porphyrin-imidazole complexes has been observed previously,81 and is a consequence of electron correlation.…”

Effects of extrinsic imidazole ligation on the molecular and electronic structure of cytochrome c

“…There is only one missing residue (Gly29). The shift of the reduced species is taken from Santos and Turner [52] b The calculated pseudocontact shift is the average of the shifts calculated over each member of the family of conformers constituting the solution structure; the standard deviation of the calculated pseudocontact shifts is also reported Figure 1 shows the short-and medium-range NOEs observed for the backbone and b-protons in the NOESY maps in H 2 O. Sequential HN-HN connectivities for stretches longer than two amino acids have been observed for residues 1±18, 20±24, 34±42, 50±56, 61±69, 85±97, and 99±103. Four prolines (residues 30, 44, 71, and 76) are present in the sequence which, together with the missing residue Gly29, break the sequential HN-HN connectivities.…”

Recent developments in the 13 C NMR spectroscopic analysis of paramagnetic hemes and heme proteins

“…Since the hyperfine shift is the sum of several contributions, including the diamagnetic shift (see Eqs. 1, 2, and 3), it is possible to obtain the 13 C contact shift if the diamagnetic contribution is known; the value of δ dia is typically obtained from the same protein in its reduced, typically low-spin S=0, diamagnetic form [121].…”