13C-NMR spectroscopy of acetyltyrosyl-guanidinated horse heart cytochrome c

Ronald A. Nieman, John Devens Gust, John R. Cronin

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The tyrosine residues of guanidinated horse heart cytochrome c have been specifically acetylated by reaction with N-[1-13C]acetylimidazole (90 atom%). Acetylation was monitored by 13C-NMR spectroscopy. The tyrosine residues were found to show widely varying reactivities ranging from one that is completely and exclusively acetylated at low reagent concentration (residue 67) to one that is acetylated only when the protein is unfolded (residue 97). Homogeneous derivatives were prepared containing one (either residue 67 or 97), three (48, 67 and 74), or four (residues 48, 67, 74 and 97) O-[1-13C]acetyl groups. 13C-NMR spectra of selected derivatives were obtained at pH 5-8, in the presence of cyanide ion, in the ferrous and ferric oxidation states, and after denaturation with 6M guanidine hydrochloride. The O-[1-13C]acetyltyrosyl resonances gave chemical shift values ranging from 171.8 to 176.0 ppm. These resonances were assigned to specific groups based on the known order of reactivity of the tyrosyl side chains toward N-acetylimidazole. The chemical shift of O-[1-13]C]acetyltyrosyl 67 was round to be particularly sensitive to changes in protein structure. The proximity of this group to the heme makes it subject to distance-dependent paramagnetic and ring current effects. Acetylation of tyrosyl 74 gives rise to a pH-dependent equilibrium between conformers in the ferric state and a conformation change in the ferrous state. Acetylation of this residue also ieads to an absorbance decrease at 695 nm that can be related to the 13C-NMR-detected conformational equilibrium. Addition of cyanide ion abolishes this equilibrium.

Original languageEnglish
Pages (from-to)144-155
Number of pages12
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume704
Issue number1
DOIs
Publication statusPublished - May 21 1982

Keywords

  • (Horse heart)
  • C-NMR
  • Acetyltyrosine
  • Cytochrome c
  • Guanidation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology
  • Medicine(all)

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