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The conformational transition of horse heart porphyrin c
Insights
Removing heme iron from cytochrome c yields porphyrin c, which retains native properties but denatures more easily. Refolding occurs in two phases, suggesting protein axial ligation influences cytochrome c folding kinetics.
Area of Science:
- Biochemistry
- Protein Chemistry
- Spectroscopy
Background:
- Cytochrome c is a vital protein in cellular respiration.
- Understanding protein folding mechanisms is crucial for comprehending biological functions and diseases.
Purpose of the Study:
- To investigate the structural and kinetic properties of porphyrin c, a heme-removed derivative of horse heart cytochrome c.
- To elucidate the folding pathway and kinetics of cytochrome c after denaturation.
Main Methods:
- Selective removal of heme iron using anhydrous hydrogen fluoride (HF).
- Characterization of porphyrin c using viscosity, far-ultraviolet circular dichroism (CD), and fluorescence spectroscopy.
- Denaturation and refolding studies using guanidine hydrochloride (GdnHCl) and heat.
- Kinetic analysis of refolding using stopped-flow absorbance and fluorescence measurements.
Main Results:
- Porphyrin c retains native-like viscosity, CD, and fluorescence properties but shows increased susceptibility to denaturation.
- Conformational transitions of porphyrin c occur reversibly at 0.95 M GdnHCl.
- Guanidine-denatured porphyrin c refolds in two kinetic phases (20 ms and 200 ms), with the faster phase accounting for ~80% of the change.
- Increased solvent viscosity did not significantly alter refolding kinetics.
Conclusions:
- The folding of guanidine-denatured cytochrome c is likely not diffusion-limited.
- Protein axial ligation appears to be a key factor driving the slower phase of cytochrome c refolding.
Abstract:
The heme iron of horse heart cytochrome c was selectively removed using anhydrous HF. The product, porphyrin c, exhibits the viscosity, far ultraviolet circular dichroic, and fluorescence properties characteristic for native cytochrome c. However, porphyrin c is more susceptible to denaturation by guanidine hydrochloride and by heat than is the parent cytochrome. All of the conformational parameters of porphyrin c exhibit a common reversible transition centered at 0.95 m guanidine hydrochloride at 23 degrees C and pH 7.0. Guanidine denatured porphyrin c refolds in two kinetic phases having time constants of 20 and 200 ms as detected by stopped flow absorbance or fluorescence measurement, with about 80% of the observed change in the faster phase. The kinetics of porphyrin c refolding are not significantly altered by increasing the viscosity of the refolding solvent 15-fold by addition of sucrose. We suggest that the folding of guanidine denatured cytochrome c is not a diffusion-limited process and that the requirement for protein axial ligation elicits the slow (s) kinetic phase observed in the refolding of cytochrome c.