Apocytochrome P450cam is a native protein with some intermediate-like properties

Area of Science:

• Biochemistry
• Protein Folding
• Enzymology

Background:

• Cytochrome P450cam is a crucial enzyme involved in various metabolic processes.
• Understanding the structural and stability dynamics of holo- and apo forms is vital for enzyme function.
• The role of the prosthetic group in protein stability is a key area of research.

Purpose of the Study:

• To investigate the structural and thermodynamic differences between holo- and apocytochrome P450cam.
• To elucidate the impact of the prosthetic group on protein stability and folding.
• To characterize the stability and intermediate-like properties of apocytochrome P450cam.

Main Methods:

• Differential scanning calorimetry (DSC) for thermal transitions.
• Limited proteolysis to assess structural integrity.
• Second-derivative spectroscopy and circular dichroism (CD) for secondary and tertiary structure analysis.
• Size-exclusion chromatography to determine protein compactness.

Main Results:

• Holocytochrome P450cam exhibits three distinct folding domains, with the prosthetic group associated with the least stable domain (41.9°C).
• Apocytochrome P450cam shows reduced helical content and a significantly lower enthalpy change upon melting (135 kJ/mol vs. 980 kJ/mol for holo form).
• Apo form displays destabilized tertiary structure, increased proteolytic susceptibility, and exhibits intermediate-like properties with very low stability (ΔG = 7.5 kJ/mol).

Conclusions:

• The prosthetic group is essential for the high stability of cytochrome P450cam.
• Apocytochrome P450cam, despite its low stability, represents a native protein with intermediate characteristics.
• These findings offer insights into the in vivo folding mechanisms and stability requirements of proteins.