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ATP specifically drives refolding of non-native conformations of cytochrome c

¹ Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma "Tor Vergata," 00133 Roma, Italy² Dipartimento di Biologia, Università Roma Tre, 00146 Roma, Italy³ Dipartimento di Chimica, Università di Firenze, 50019 Sesto Fiorentino, Italy

(RECEIVED September 6, 2004; FINAL REVISION November 19, 2004; ACCEPTED January 7, 2005)

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.

Keywords: cytochrome c; oleic acid; molten globule; nucleotides; resonance Raman spectroscopy

Abbreviations: cyt c, horse heart cytochrome c • cmc, critical micelle concentration • LS, low spin • HS, high-spin • CD, circular dichroism • RR, resonance Raman

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041069405.

Reprint requests to: Laura Fiorucci, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma "Tor Vergata," via Montpellier 1, 00133 Roma, Italy; e-mail: fiorucci{at}uniroma2.it; fax: +39-0672596477.

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