Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Cytochrome P450cam (P450cam) crystal structures with putidaredoxin (Pdx) show an open conformation.
• Pdx binding is hypothesized to free Asp251 for O2 activation via a proton relay network.
• The open state's looser substrate interactions conflict with observed hydroxylation selectivity.

Purpose of the Study:

• To investigate the conformational changes induced by Pdx binding in P450cam.
• To reconcile the open conformation with substrate binding and catalytic activity.
• To elucidate the role of Asp251 in Pdx-dependent catalysis.

Main Methods:

• Molecular dynamics simulations of P450cam-Pdx interactions.
• Enzyme activity assays of wild-type and R186A mutant P450cam.
• X-ray crystallography of P450cam with bound product.

Main Results:

• Pdx binding favors a conformation that stabilizes the active site and reduces camphor mobility.
• A partially open conformation compatible with the proton relay network is maintained.
• The R186A mutant shows activity without Pdx, indicating Asp251 release is key.
• X-ray structure confirms product binding in the R186A mutant.

Conclusions:

• Pdx binding stabilizes the P450cam active site while facilitating proton relay for O2 activation.
• Asp251 release from salt bridges is crucial for P450cam function.
• These findings support Pdx's role in enabling proton-coupled electron transfer in P450cam catalysis.