Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Area of Science:

• Biochemistry
• Computational Biology
• Structural Biology

Background:

• Cytochrome P450cam (CYP101) is a crucial enzyme involved in drug metabolism and xenobiotic degradation.
• Understanding water dynamics within the active site is key to elucidating enzyme function and substrate binding.
• Previous studies relied on crystal structures, offering static snapshots of the active site.

Purpose of the Study:

• To investigate the dynamic behavior of water molecules in the active site of both apo- and camphor-bound cytochrome P450cam.
• To identify and characterize pathways for water diffusion into and out of the protein.
• To understand how substrate binding influences active site hydration.

Main Methods:

• Long-timescale molecular dynamics simulations (300 ns) were performed on apo- and camphor-bound CYP101.
• Potential-of-mean-force calculations were used to identify water diffusion pathways.
• No biased sampling methods were employed to ensure natural water movement.

Main Results:

• Apo-CYP101 active site accommodates an average of 6.4 water molecules, with up to 12 observed, showing rapid hydration and exchange with bulk solvent.
• Camphor-bound CYP101 shows zero water molecules in the active site, consistent with crystal structures.
• Thermodynamically favored trans-protein pathways for water diffusion were identified.
• Camphor binding alters the free-energy landscape, promoting water efflux from the active site.

Conclusions:

• The active site of cytochrome P450cam is highly dynamic regarding water content, with significant hydration in the absence of substrate.
• Substrate binding effectively blocks water ingress and facilitates its egress, crucial for enzyme activity.
• Specific pathways govern water exchange, and their energetics are modulated by substrate presence.