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Protein stability and mutations in the axial methionine loop of a minimal cytochrome c

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Abstract

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

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Abbreviations

Bpcytc :

soluble fragment of cytochrome c 553 from Bacillus pasteurii

GdmCl:

guanidinium chloride

I75A:

Ile75 to Ala mutant

I75V:

Ile75 to Val mutant

P72A:

Pro72 to Ala mutant

P72G:

Pro72 to Gly mutant

Q68K:

Gln75 to Lys mutant

WT:

wild type

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Acknowledgements

Financial support from MIUR is gratefully acknowledged. P.R.V. thanks the European Commission for financial support under contract number HPRN-CT-2000-00092.

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Authors and Affiliations

  1. Magnetic Resonance Center (CERM), University of Florence, Via L. Sacconi 6, 50019 , Sesto Fiorentino, Italy

    Ilaria Bartalesi, Ivano Bertini, Antonio Rosato, Murugendra Vanarotti, Paul R. Vasos & Maria Silvia Viezzoli

  2. Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 , Sesto Fiorentino, Italy

    Ivano Bertini, Antonio Rosato & Maria Silvia Viezzoli

  3. Department of Chemistry, University of Modena and Reggio Emilia, Via G. Campi 183, 41100 , Modena, Italy

    Giulia Di Rocco & Antonio Ranieri

Authors
  1. Ilaria Bartalesi
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  2. Ivano Bertini
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  3. Giulia Di Rocco
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  4. Antonio Ranieri
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  5. Antonio Rosato
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  6. Murugendra Vanarotti
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  7. Paul R. Vasos
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  8. Maria Silvia Viezzoli
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Correspondence to Ivano Bertini.

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Bartalesi, I., Bertini, I., Di Rocco, G. et al. Protein stability and mutations in the axial methionine loop of a minimal cytochrome c . J Biol Inorg Chem 9, 600–608 (2004). https://doi.org/10.1007/s00775-004-0558-8

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  • DOI: https://doi.org/10.1007/s00775-004-0558-8

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