<sup>13</sup>C‐NMR studies of horse ferrocytochrome <i>c</i>

Santos, Helena; Turner, David L.

doi:10.1016/0014-5793(85)80614-1

Cited by 12 publications

(7 citation statements)

References 21 publications

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“…1. The 55 methyl groups, including six from the heme and its thioether bridges, resonate in the region 9-28 ppm [4] The 13C spectrum of the oxidised protein is expected to be broadly similar, with pseudocontact shifts of less than 2 ppm for most of the residues. The haem and the axial ligands, however, should experience Fermi contact shifts through interaction with the delocalised unpaired electron.…”

Section: Resultsmentioning

confidence: 99%

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¹³C and proton NMR studies of horse cytochrome c

Santos

Turner

1992

European Journal of Biochemistry

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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...

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Section: Resultsmentioning

confidence: 99%

“…Recent work on horse ferrocytochrome c by Gao et al [lo] has confirmed most of the existing assignments [4] and added a number of assignments for c1 carbons. We now report the full set of 13C assignments which are available for both oxidation states, including a number of additional contact-shifted resonances, together with their temperature dependences.…”

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confidence: 92%

¹³C and proton NMR studies of horse cytochrome c

Santos

Turner

1992

European Journal of Biochemistry

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“…These pioneering experiments, which permitted the observation of 13 C resonances from heme vinyl groups in high-spin and low-spin myoglobin derivatives [39], demonstrated the practicality and importance of applying 13 C NMR spectroscopy to the analysis of paramagnetic heme proteins. More recently, the 1 H-13 C COSY spectra of natural abundance ferricytochrome c [49] and that of sperm whale myoglobin [50], were utilized to assign the 13 C resonances originating from the heme methyl groups. Subsequently, the proton-detected heteronuclear multiple quantum coherence (HMQC) experiment [51] was utilized to identify several heme carbons and their corresponding proton resonances in the paramagnetic active site of cytochrome c 550 [52], which culminated in the…”

Section: Mario Rivera · Gregori a Caignanmentioning

confidence: 99%

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

Rivera

Caignan

2004

Analytical and Bioanalytical Chemistry

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Despite the wealth of information that has been obtained from the study of paramagnetic hemes and heme proteins by 1H NMR spectroscopy, there are certain limitations imposed by the nature of paramagnetically affected resonances that are difficult to overcome. Although it has long been recognized that 13C NMR spectroscopy is likely to be a powerful complementary technique to overcome some of these limitations, the low sensitivity and low natural abundance of 13C nuclei has resulted in a lag in the application of 13C NMR spectroscopy to the study of paramagnetic hemes and heme proteins. The tremendous advances in methodology and instrumentation witnessed in the NMR field, coupled to the advent of recombinant DNA methods that have made possible the preparation and purification of significant quantities of proteins, and the biosynthesis of 13C-labeled heme, have contributed to an increased interest in the study of paramagnetic heme active sites by 13C NMR spectroscopy. As a consequence, 13C NMR spectroscopy is emerging as a powerful tool to study heme electronic structure and structure-function relationships in heme-containing proteins. In this report we strive to summarize some of the recent developments in the analysis of paramagnetic hemes and heme-containing proteins by 13C NMR spectroscopy.

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“…capsulatus (Wand et al, 1989;Gao et al, 1990a;Marion and Guerlesquin, 1992;Chau et al, 1990;Detlefsen et al, 1990;Cai et al, 1992;. In addition, assignments for the 13C of horse cytochrome c (Santos and Turner, 1985, Correspondence to D. Marion Gao et al, 1990b) and D. vulgaris cytochrome c-553 (Medvedeva et a]., 1993) have been obtained at natural abundance. Moreover, the NMR-derived solution structures have been determined for the cytochromes c from Ps.…”

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Assignment of the ¹³C and ¹³CO resonances for Rhodobacter capsulatus ferrocytochrome c₂ using double‐resonance and triple‐resonance NMR spectroscopy

Caffrey

Brutscher

Simorre

et al. 1994

European Journal of Biochemistry

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Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two‐ and three‐dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13Cα and 13CO secondary shifts were better indicators of secondary structure than 1Hα and 13Cβ secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13Cα and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.

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¹³C‐NMR studies of horse ferrocytochrome c

Cited by 12 publications

References 21 publications

¹³C and proton NMR studies of horse cytochrome c

¹³C and proton NMR studies of horse cytochrome c

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

Assignment of the ¹³C and ¹³CO resonances for Rhodobacter capsulatus ferrocytochrome c₂ using double‐resonance and triple‐resonance NMR spectroscopy

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13C‐NMR studies of horse ferrocytochrome c

Cited by 12 publications

References 21 publications

13C and proton NMR studies of horse cytochrome c

13C and proton NMR studies of horse cytochrome c

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

Assignment of the 13C and 13CO resonances for Rhodobacter capsulatus ferrocytochrome c2 using double‐resonance and triple‐resonance NMR spectroscopy

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¹³C‐NMR studies of horse ferrocytochrome c

¹³C and proton NMR studies of horse cytochrome c

¹³C and proton NMR studies of horse cytochrome c

Assignment of the ¹³C and ¹³CO resonances for Rhodobacter capsulatus ferrocytochrome c₂ using double‐resonance and triple‐resonance NMR spectroscopy