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The subunit location of magnesium in cytochrome c oxidase
1Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Insights
Researchers successfully removed magnesium ions from bovine heart cytochrome c oxidase, preserving enzyme function. This magnesium depletion reveals a key binding site on subunit IV, crucial for understanding enzyme activity.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein chemistry
Background:
- Bovine heart cytochrome c oxidase is a crucial enzyme in cellular respiration.
- The role of magnesium ions in the structure and function of this enzyme is not fully understood.
- Previous methods for magnesium depletion often led to protein denaturation.
Purpose of the Study:
- To develop a method for selectively depleting magnesium ions from bovine heart cytochrome c oxidase without causing denaturation.
- To investigate the location of magnesium ions within the enzyme structure.
- To assess the impact of magnesium ions on enzyme activity.
Main Methods:
- Heat treatment of bovine heart cytochrome c oxidase at 43°C followed by dialysis against EDTA buffer solution.
- Activity assays of native, heat-treated, and magnesium-depleted enzymes.
- Labeling of exposed carboxyl groups on subunit IV using N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide (NCD-4).
Main Results:
- Up to 75% magnesium depletion was achieved without significant protein denaturation.
- The magnesium-depleted enzyme retained 40% of the native enzyme's activity.
- Magnesium depletion exposed a carboxyl group on subunit IV, indicated by enhanced NCD-4 labeling, suggesting magnesium's location and ligand interactions.
Conclusions:
- A novel method for magnesium depletion from bovine heart cytochrome c oxidase without denaturation has been established.
- Magnesium ions are located in subunit IV of the enzyme, interacting with at least one carboxyl group.
- Divalent magnesium plays a role in maintaining the full activity of bovine heart cytochrome c oxidase.
Abstract:
The magnesium ion in bovine heart cytochrome c oxidase can be depleted up to 75% by heat treatment of the enzyme at 43 degrees C followed by dialysis against EDTA buffer solution. The magnesium-depleted enzyme so obtained retains 40% of the activity of the native enzyme. This is the first attempt to deplete magnesium ion from bovine heart cytochrome c oxidase without denaturation of the protein. Magnesium depletion exposes at least one carboxyl group on subunit IV for labeling by N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide (NCD-4). The NCD-4 labeling of subunit IV of the magnesium-depleted enzyme is significantly enhanced relative to what is observed for the native and heat-treated oxidase, suggesting that the magnesium ion is located in subunit IV with at least one carboxyl ligand. By comparing the activity of the magnesium-depleted enzyme with that of a control sample of heat-treated oxidase, the influence of divalent magnesium on the activity of the enzyme is assessed.