ATP and ADP bind to cytochrome c oxidase and regulate its activity

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein structure

Background:

• Cytochrome c oxidase (COX) is a key enzyme in cellular respiration.
• Understanding nucleotide binding and its effects on COX is crucial for elucidating energy metabolism.
• Previous studies have hinted at regulatory roles for nucleotides in COX function.

Purpose of the Study:

• To quantify ATP and ADP binding sites on bovine heart cytochrome c oxidase.
• To investigate the functional and conformational consequences of nucleotide binding.
• To identify the specific subunit involved in nucleotide-mediated regulation.

Main Methods:

• Equilibrium dialysis using radiolabeled ATP and ADP analogs.
• Filtration assays to assess time-dependent binding.
• Spectroscopic analysis (gamma-band changes) to detect conformational shifts.
• Polarographic measurements of enzyme activity.
• Enzyme inhibition studies using monoclonal antibodies against subunit IV.

Main Results:

• Seven ATP and ten ADP binding sites were identified per monomer of isolated COX.
• ATP binding is time-dependent, linked to cholate dissociation.
• ADP binding induces a 12% decrease in the gamma-band of Tween 20-solubilized COX, indicating conformational changes specific to ADP.
• COX activity is inhibited by high ATP/ADP ratios in Tween 20, an effect reversed by anti-subunit IV antibody.
• Dodecylmaltoside-solubilized COX shows higher activity, unaffected by nucleotide preincubation.

Conclusions:

• Bovine heart COX possesses multiple specific binding sites for ATP and ADP.
• ADP binding induces specific conformational changes in certain COX preparations.
• Nucleotide regulation of COX activity involves interaction with subunit IV, particularly in Tween 20-solubilized forms.
• Detergent choice significantly impacts COX activity and its response to nucleotides.