Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.
Abstract: One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the holoprotein. These results indicate that, at neutral pH, the ligation of His18 of the iron is important for the maintenance of the native structure whereas the Met80 ligation is not essential, and that porphyrin-cytochrome c assumes a molten globule-like state. Porphyrin-cytochrome c was largely unfolded at pH 2.0 in the absence of salt, but assumed another molten globule-like structure in the presence of anions. The salt-induced stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than holocytochrome c. These results indicate that, at acidic pH, the His18 ligation is important, although not essential, for stabilizing the molten globule state. Taken together, both specific (i.e. the His18 axial ligand, as observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state.
Publication Date: 1996-02-16 PubMed ID: 8609608DOI: 10.1006/jmbi.1996.0075Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article focuses on determining the role of heme axial ligands in maintaining the structural stability of the horse cytochrome c protein in both its native and molten globule states, using an iron-free version of the cytochrome c protein for comparison. It concludes that certain molecular interactions, such as those with the His18 axial ligand and the heme, are crucial for the protein’s stability.
Research Methodology and Findings
- The researchers first examined the behavior of porphyrin-cytochrome c, an iron-free version of horse cytochrome c, at neutral pH.
- Using far-UV circular dichroism, they concluded that the protein had native-like alpha-helices, yet the near-UV CD suggested flexibility in the side chains.
- They found that the protein’s ability to resist heat or denaturants was weaker than that of the intact protein. This stability level is close to that of the acidic molten globule state of the holoprotein (the protein with its integral heme group).
- These findings led them to conclude that the ligation of His18 of the iron is key to maintaining the native protein structure, while Met80 ligation is not. It also suggests that porphyrin-cytochrome c assumes a molten globule state at a neutral pH.
Further Experiments and Conclusions
- The next step for the research team was to examine the behavior of porphyrin-cytochrome c at acidic pH (pH 2.0), both with and without the presence of salt. In the absence of salt, they found that it unfolded largely but produced another molten globule-like structure when salt was present.
- However, higher concentrations of salt were necessary to stabilize this molten globule-like state compared to the holocytochrome c. It concluded that His18 ligation is crucial, though not essential, for maintaining this molten globule state under acidic conditions.
- In conclusion, this work indicates that both specific interactions (like the one with His18 axial ligand under acidic conditions) and non-specific interactions (like the hydrophobic effects of the heme at neutral pH) play role in maintaining stability of the molten globule state of horse cytochrome c protein.
Cite This Article
APA
Hamada D, Kuroda Y, Kataoka M, Aimoto S, Yoshimura T, Goto Y.
(1996).
Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.
J Mol Biol, 256(1), 172-186.
https://doi.org/10.1006/jmbi.1996.0075 Publication
Researcher Affiliations
- Department of Biology Faculty of Science Osaka University, Japan.
MeSH Terms
- Animals
- Cytochrome c Group / chemistry
- Heme / chemistry
- Horses
- Hydrogen-Ion Concentration
- Ligands
- Magnetic Resonance Spectroscopy
- Models, Molecular
- Molecular Structure
- Porphyrins / chemistry
- Protein Conformation
- Protein Denaturation
- Protein Folding
- Protein Structure, Secondary
- Protein Structure, Tertiary
- Scattering, Radiation
- Spectrometry, Fluorescence
- Thermodynamics
- X-Rays
Citations
This article has been cited 11 times.- Parray ZA, Shahid M, Islam A. Insights into Fluctuations of Structure of Proteins: Significance of Intermediary States in Regulating Biological Functions. Polymers (Basel) 2022 Apr 11;14(8).
- Fonin AV, Darling AL, Kuznetsova IM, Turoverov KK, Uversky VN. Intrinsically disordered proteins in crowded milieu: when chaos prevails within the cellular gumbo. Cell Mol Life Sci 2018 Nov;75(21):3907-3929.
- Pawlowska R, Janicka M, Jedrzejczyk D, Chworos A. RNA fragments mimicking tRNA analogs interact with cytochrome c. Mol Biol Rep 2016 Apr;43(4):295-304.
- Yamanaka M, Nagao S, Komori H, Higuchi Y, Hirota S. Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping. Protein Sci 2015 Mar;24(3):366-75.
- Tsai MY, Yuan JM, Teranishi Y, Lin SH. Thermodynamics of protein folding using a modified Wako-Saitô-Muñoz-Eaton model. J Biol Phys 2012 Sep;38(4):543-71.
- Hamaguchi M, Kamikubo H, Suzuki KN, Hagihara Y, Yanagihara I, Sakata I, Kataoka M, Hamada D. Structural basis of α-catenin recognition by EspB from enterohaemorrhagic E. coli based on hybrid strategy using low-resolution structural and protein dissection. PLoS One 2013;8(8):e71618.
- Alam Khan MK, Rahaman MH, Hassan MI, Singh TP, Moosavi-Movahedi AA, Ahmad F. Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c. J Biol Inorg Chem 2010 Nov;15(8):1319-29.
- Alam Khan MK, Das U, Rahaman MH, Hassan MI, Srinivasan A, Singh TP, Ahmad F. A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0. J Biol Inorg Chem 2009 Jun;14(5):751-60.
- Russell BS, Melenkivitz R, Bren KL. NMR investigation of ferricytochrome c unfolding: detection of an equilibrium unfolding intermediate and residual structure in the denatured state. Proc Natl Acad Sci U S A 2000 Jul 18;97(15):8312-7.
- Kataoka M, Kuwajima K, Tokunaga F, Goto Y. Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering. Protein Sci 1997 Feb;6(2):422-30.
- Sebastiani F, Bocedi A, De Simone G, Notari S, Gambardella G, Sbardella D, Smulevich G, Ascenzi P, Coletta M. Proton-linked effects on the axial coordination in ferric and ferrous horse heart cytochrome c. Protein Sci 2025 Nov;34(11):e70233.
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