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The 40s Ω-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c

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The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1–38 heme-containing N-fragment with the residues 57–104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega (Ω)-loop region. The fragment complex shows compact structure, native-like α-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1–38/57–104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (pKa1=6.72) and a high pKa-associated state transition (pKa2=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (pKa=8.9). The data indicate that the 40s Ω-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.

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Abbreviations

CD:

circular dichroism

CV:

cyclic voltammetry

cyt c:

cytochrome c

RR:

resonance Raman

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Acknowledgements

The authors wish to thank P. Sarti and E. Forte (Dipartimento di Biochimica, Università di Roma “La Sapienza”) for assistance in the polarographic measurements, and E. Schininà (Dipartimento di Biochimica, Università di Roma “La Sapienza”) for mass spectrometric measurements. This research was funded in part by grants from the Italian MIUR (COFIN 2001 031798).

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Authors and Affiliations

  1. Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma “Tor Vergata”, via Montpellier 1, 00133 , Rome, Italy

    Paola Caroppi, Federica Sinibaldi, Laura Fiorucci, Franca Ascoli & Roberto Santucci

  2. Dipartimento di Chimica, Università di Firenze, via della Lastruccia 3, 50019 , Sesto Fiorentino, Italy

    Elisa Santoni, Barry D. Howes & Giulietta Smulevich

  3. Dipartimento di Chimica, Università di Roma “La Sapienza”, P. le A. Moro 5, 00185 , Rome, Italy

    Tommaso Ferri

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  1. Paola Caroppi
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  2. Federica Sinibaldi
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  3. Elisa Santoni
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  4. Barry D. Howes
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  5. Laura Fiorucci
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  8. Giulietta Smulevich
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  9. Roberto Santucci
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Correspondence to Roberto Santucci.

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Caroppi, P., Sinibaldi, F., Santoni, E. et al. The 40s Ω-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c. J Biol Inorg Chem 9, 997–1006 (2004). https://doi.org/10.1007/s00775-004-0601-9

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  • DOI: https://doi.org/10.1007/s00775-004-0601-9

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