ATP specifically drives refolding of non-native conformations of cytochrome c.
An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.
Sinibaldi
Federica
F
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata, via Montpellier 1, 00133 Rome, Italy.
Mei
Giampiero
G
Polticelli
Fabio
F
Piro
M Cristina
MC
Howes
Barry D
BD
Smulevich
Giulietta
G
Santucci
Roberto
R
Ascoli
Franca
F
Fiorucci
Laura
L
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D009711:Nucleotides / Q000494:pharmacology; D019301:Oleic Acid / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D013059:Spectrum Analysis, Raman
D009711:Nucleotides; D019301:Oleic Acid; D000255:Adenosine Triphosphate; D045304:Cytochromes c
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