His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c. Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability. The His26Tyr mutant shows properties typical of the molten globule. Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein. The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein. Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine. Furthermore, a reduction in catalytic activity indicates that the functional properties are altered. The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44. Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein. In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH. Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect. This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80. By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant. This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c.

Sinibaldi, F., Piro, M.c., Howes, B.d., Smulevich, G., Ascoli, F., Santucci, R. (2003). Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c, 42(24), 7604-7610 [10.1021/bi034132r].

Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c

SINIBALDI, FEDERICA;PIRO, MARIA CRISTINA;SANTUCCI, ROBERTO
2003-01-01

Abstract

His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c. Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability. The His26Tyr mutant shows properties typical of the molten globule. Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein. The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein. Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine. Furthermore, a reduction in catalytic activity indicates that the functional properties are altered. The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44. Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein. In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH. Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect. This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80. By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant. This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c.
2003
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
Catalysis; Hydrogen bonds; Mutagenesis; pH effects; Spectroscopic analysis; Yeast; Intramolecular bonds; Cytology; cytochrome c; heme iron; histidine; iron derivative; tyrosine; unclassified drug; article; catalysis; circular dichroism; controlled study; hydrogen bond; ligand binding; macromolecule; measurement; nonhuman; pH; priority journal; protein binding; protein conformation; protein expression; protein function; protein localization; protein stability; protein structure; spectroscopy; Amino Acid Substitution; Circular Dichroism; Cytochrome c Group; Electrochemistry; Guanidine; Heme; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Protein Denaturation; Protein Folding; Recombinant Proteins; Spectrophotometry; Spectrum Analysis, Raman; Thermodynamics; Yeasts
Sinibaldi, F., Piro, M.c., Howes, B.d., Smulevich, G., Ascoli, F., Santucci, R. (2003). Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c, 42(24), 7604-7610 [10.1021/bi034132r].
Sinibaldi, F; Piro, Mc; Howes, Bd; Smulevich, G; Ascoli, F; Santucci, R
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/49761
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