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Autoxidizability of beef heart cytochrome c1 lacking the hinge protein c1-c
Insights
Stable binding of the hinge protein to the heme subunit is essential for preventing autoxidation in beef heart cytochrome c1. This interaction, involving cysteinyl residues, maintains the protein
Area of Science:
- Biochemistry
- Protein Structure and Function
- Electron Transport Chain
Background:
- Beef heart cytochrome c1 is a crucial component of the electron transport chain.
- Its autoxidizability, or tendency to react with oxygen, is a key characteristic influencing its function.
- The interaction between the heme subunit and the hinge protein is thought to regulate this property.
Purpose of the Study:
- To investigate the role of the hinge protein's binding to the heme subunit in the autoxidizability of beef heart cytochrome c1.
- To determine if the integrity of this protein-protein interaction is essential for preventing heme autoxidation.
Main Methods:
- Isolation of cytochrome c1 as a heme subunit-hinge protein subcomplex.
- Treatment with p-chloromercuribenzoate (pCMB) to alter subunit binding.
- Analysis using polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography.
- Assessment of autoxidizability before and after chemical treatments and subunit manipulation.
Main Results:
- pCMB treatment weakened the binding between the heme and hinge subunits, leading to slight autoxidizability.
- Reversal of pCMB binding with 2-mercaptoethanol restored native binding and repressed autoxidizability.
- Isolated heme subunits, even after pCMB removal, exhibited significant autoxidizability, which was repressed by cholate.
Conclusions:
- The stable binding of the hinge protein to the heme subunit is essential for the nonautoxidizability of the cytochrome c1 subcomplex.
- Cysteinyl residues within the subcomplex are likely involved in mediating this stable subunit interaction.
- Disruption of this binding exposes the heme to oxidation.
Abstract:
The autoxidizability of beef heart cytochrome c1 was investigated in terms of the integrity of the binding of the hinge protein to the heme subunit. Cytochrome c1 was isolated as a subcomplex consisting of the heme subunit and the hinge protein. Treatment of the cytochrome c1 subcomplex with p-chloromercuribenzoate (pCMB) under mild conditions lessened the binding strength between the two subunits. They were dissociated on polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions, but were not separated by gel filtration chromatography. The pCMB-treated subcomplex had a slight autoxidizability. This was repressed to the level of the native subcomplex, when the mercurial compound bound to the subcomplex was removed by the addition of 2-mercaptoethanol. Concomitantly, the less stable binding between the subunits was apparently reversed to the native state. After pCMB treatment of the subcomplex, the heme subunit recovered from PAGE showed marked autoxidizability, even if it was treated with 2-mercaptoethanol. Addition of cholate repressed the autoxidizability of the heme subunit after the removal of the mercurial compound. These results confirmed that the stable binding of the hinge protein to the heme subunit was essential for the nonautoxidizability of cytochrome c1 subcomplex. In addition, it was suggested that cysteinyl residues in the subcomplex must be involved to a great extent in the stable binding between the two subunits.