Disentangling Ligand Migration and Heme Pocket Relaxation
in Cytochrome P450cam
CATHERINE TETREAU 1, LILIANE MOUAWAD 1, SAMUEL MURAIL 1, PATRICIA DUCHAMBON 1, YVES BLOUQUIT 1 and Daniel LAVALETTE 1*
1 Biophysique Moléculaire, Institut Curie, Centre Universitaire, 91405 Orsay, France
* To whom correspondence should be addressed. E-mail: daniel.lavalette{at}curie.u-psud.fr.
Submitted on July 20, 2004
Revised on September 3, 2004
Accepted on 14 September 2004
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Abstract |
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In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of CO rebinding with cytochrome P450cam display up to 5 distinct processes between 77 K and 300 K. They were disentangled by using a combination of three approaches: i) the competition of the ligand with xenon for the occupation of internal protein cavities; ii) the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; iii) Molecular Mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek for internal cavities. In cytochrome P450cam, active site conformational relaxation results from the displacement of the substrate towards the heme center upon photodissociation of the ligand. It is responsible for the long puzzling bimodal nature of the rebinding kinetics observed down to 77 K. The relaxation rate is strongly substrate dependent. Ligand migration is slower and is observed only above 135 K. Migration and return rates are independent of the substrate.
Key Words:
Cavities in P450cam, Laser photolysis, Molecular mechanics, Protein dynamics, Rebinding Kinetics, Xenon binding
