The subunit structure of Pseudomonas cytochrome oxidase

Area of Science:

• Biochemistry
• Enzymology
• Protein chemistry

Background:

• Pseudomonas cytochrome oxidase is a key enzyme in bacterial respiration.
• The enzyme is a dimer, with each subunit containing two hemes and a molecular weight of approximately 63,000.
• Understanding the enzyme's quaternary structure stability is crucial for elucidating its function.

Purpose of the Study:

• To investigate the stability of the dimeric structure of Pseudomonas cytochrome oxidase.
• To determine the conditions that lead to the dissociation of the enzyme into subunits.

Main Methods:

• Progressive succinylation of lysine residues to modify protein structure.
• pH manipulation to assess enzyme stability.
• Sedimentation velocity analysis (S20,W) to characterize protein forms.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weights.

Main Results:

• Succinylation of 14-68% of lysine residues increased the proportion of the enzyme in subunit form (S20,W ~4 S) from 18% to 92%.
• High succinylation levels and extreme pH (below 4 or above 11) induced the formation of a 2 S component.
• SDS-PAGE confirmed a molecular weight of 63,000 for both 4 S and 2 S components, indicating subunit dissociation.
• The enzyme remained undissociated in 3 M NaCl, 1 M Na2SO4, or 6 M urea, with minor sedimentation coefficient changes attributed to preferential hydration.

Conclusions:

• The dimeric structure of Pseudomonas cytochrome oxidase is sensitive to chemical modification (succinylation) and pH extremes.
• Dissociation occurs into subunits of approximately 63,000 molecular weight, forming distinct sedimenting species (4 S and 2 S).
• The enzyme exhibits stability in high salt concentrations and urea, suggesting the involvement of non-covalent interactions sensitive to pH and chemical modification in maintaining the dimeric structure.