Kinetic barriers to the folding of horse cytochrome C in the reduced state.
Abstract: To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor.
Publication Date: 2002-10-16 PubMed ID: 12379125DOI: 10.1021/bi0204443Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research explores the kinetic barriers in the folding of horse cytochrome c using a derivative called carbonmonoxycytochrome c and finds significant differences in the folding-unfolding kinetics between the two proteins. The results suggest coordination contact formations and disruptions pose high-energy kinetic barriers to ferrocytochrome c folding and unfolding.
Methodology
- The key method used in the study was the preparation of a CO-liganded derivative of cytochrome c, titled carbonmonoxycytochrome c. This was achieved through the utilization of the thermodynamic reversibility of ferrocytochrome c unfolding caused by guanidinium hydrochloride (GdnHCl) at pH 7.
- 13C NMR and optical spectroscopy were used to study the CO binding properties of unfolded ferrocytochrome c. These were found to be similar to those of native myoglobin and isolated chains of human hemoglobin.
Findings
- By observing equilibrium unfolding transitions of ferrocytochrome c in the presence and absence of CO, it was found that no accumulation of structural intermediates occurred to a detectable level.
- The thermodynamic parameters gained through the two-state analysis of fluorescence transitions varied based on the presence of CO.
- The native states of ferrocytochrome c and carbonmonoxycytochrome c were discovered to be nearly identical in terms of structure and confirmation, the only difference being the Fe2+-M80 –> Fe2+-CO replacement.
- Significant differences were found in the folding and unfolding kinetic reactions of the two proteins when functioning as a result of GdnHCl. The folding and unfolding rates were studied through stopped-flow fluorescence.
Conclusion
- The study found that the formation and disruption of the Fe2+-M80 coordination contact imposed high-energy kinetic barriers to the folding and unfolding of ferrocytochrome c.
- The research also showed that the unfolding barrier created by the Fe2+-M80 bond provided enough kinetic stability to the native state of ferrocytochrome c to enable it to perform its physiological function as an electron donor.
Cite This Article
APA
Bhuyan AK, Kumar R.
(2002).
Kinetic barriers to the folding of horse cytochrome C in the reduced state.
Biochemistry, 41(42), 12821-12834.
https://doi.org/10.1021/bi0204443 Publication
Researcher Affiliations
- School of Chemistry, University of Hyderabad, Hyderabad 500046, India. akbsc@uohyd.ernet.in
MeSH Terms
- Animals
- Binding Sites
- Carbon Monoxide / chemistry
- Cytochrome c Group / chemistry
- Enzyme Stability
- Ferrous Compounds / chemistry
- Guanidine / chemistry
- Histidine / chemistry
- Horses
- Hydrogen-Ion Concentration
- Kinetics
- Ligands
- Methionine / chemistry
- Models, Chemical
- Nuclear Magnetic Resonance, Biomolecular
- Oxidation-Reduction
- Protein Denaturation
- Protein Folding
- Spectrometry, Fluorescence
- Spectrophotometry, Ultraviolet
Citations
This article has been cited 6 times.- Li J, Li H. New insights into the folding-unfolding mechanism and conformations of cytochrome C.. Chem Sci 2022 Jun 29;13(25):7498-7508.
- Osawa K, Kossowska D, Park K, Kwak K, Cho M. Two-dimensional infrared spectroscopic study of cytochrome c peroxidase activity in deep eutectic solvent.. Struct Dyn 2019 Nov;6(6):064703.
- Latypov RF, Maki K, Cheng H, Luck SD, Roder H. Folding mechanism of reduced Cytochrome c: equilibrium and kinetic properties in the presence of carbon monoxide.. J Mol Biol 2008 Nov 7;383(2):437-53.
- Droghetti E, Oellerich S, Hildebrandt P, Smulevich G. Heme coordination states of unfolded ferrous cytochrome C.. Biophys J 2006 Oct 15;91(8):3022-31.
- Kumar R, Prabhu NP, Yadaiah M, Bhuyan AK. Protein stiffening and entropic stabilization in the subdenaturing limit of guanidine hydrochloride.. Biophys J 2004 Oct;87(4):2656-62.
- Miksovská J, Larsen RW. Photothermal studies of pH induced unfolding of apomyoglobin.. J Protein Chem 2003 May;22(4):387-94.
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