Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration

Yash P. Myer, Swatantar Kumar, Kathleen Kinnally, Jayanti Pande

Research output: Contribution to journalArticle

Abstract

The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the FIII and FII CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pKa values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of FII, with FIII being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, FII CT-cytochrome c being the most deranged.

Original languageEnglish (US)
Pages (from-to)321-342
Number of pages22
JournalJournal of Protein Chemistry
Volume6
Issue number4
DOIs
StatePublished - Aug 1987

Fingerprint

Cytochromes c
Heme
Methionine
Cytochromes
Horses
Conformations
Proteins
sulfoxide
Dimethyl Sulfoxide
Protons
Hot Temperature
Raman Spectrum Analysis
Circular Dichroism
Sulfur
Isomers
Hydroxyl Radical
Ionization
Raman spectroscopy
Thermodynamic stability
Magnetic Resonance Spectroscopy

Keywords

  • conformation
  • CT-cytochromes
  • cytochrome c
  • heme configuration

ASJC Scopus subject areas

  • Biochemistry

Cite this

Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration. / Myer, Yash P.; Kumar, Swatantar; Kinnally, Kathleen; Pande, Jayanti.

In: Journal of Protein Chemistry, Vol. 6, No. 4, 08.1987, p. 321-342.

Research output: Contribution to journalArticle

Myer, Yash P. ; Kumar, Swatantar ; Kinnally, Kathleen ; Pande, Jayanti. / Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration. In: Journal of Protein Chemistry. 1987 ; Vol. 6, No. 4. pp. 321-342.
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abstract = "The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the FIII and FII CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pKa values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of FII, with FIII being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, FII CT-cytochrome c being the most deranged.",
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