The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 2. Conformational analysis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Adrenal mitochondrial cholesterol-side-chain-cleaving cytochrome P450 is crucial for steroidogenesis.
• Previous thermodynamic analysis indicated spin-state equilibrium related to enzyme conformation.
• Structural confirmation of these conformational changes is needed.

Purpose of the Study:

• To structurally characterize the enzyme conformational changes of cholesterol-side-chain-cleaving cytochrome P450.
• To correlate these changes with spin-state equilibrium and thermodynamic data.
• To elucidate the impact of pH and temperature on enzyme conformation.

Main Methods:

• Kinetic analysis of spin-transition following rapid pH jumps in aqueous buffer and ethylene glycol.
• Measurement of activation energy (Ea) for spin-transition kinetics.
• Circular Dichroism (CD) spectroscopy to monitor pH and temperature effects on enzyme conformation.

Main Results:

• Spin-transition kinetics were multiphasic in buffer and biphasic in 35% ethylene glycol.
• Exceptionally high activation energy (147 kJ.mol-1) suggests large-scale conformational changes.
• CD spectra revealed an equilibrium between at least two conformations, independent of spin state.
• High-temperature conformation showed decreased tyrosine environment polarity and a 4% decrease in helical content.

Conclusions:

• Structural evidence supports the existence of distinct enzyme conformations in equilibrium.
• These conformational changes are linked to significant protein structural rearrangements.
• The findings reconcile thermodynamic data with structural observations of cytochrome P450.