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Substrate binding favors enhanced NO binding to P450cam

Item Type:Article
Title:Substrate binding favors enhanced NO binding to P450cam
Creators Name:Franke, A. and Stochel, G. and Jung, C. and van Eldik, R.
Abstract:Ferric cytochrome P450cam from Pseudomonas putida (P450 cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO). The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly. In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate. The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure. Large and positive values of ΔS‡ and ΔV‡ observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO. In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process. Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level. Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects. The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.
Keywords:Camphor, Camphor 5-Monooxygenase, Hydrogen-Ion Concentration, Kinetics, Nitric Oxide, Substrate Specificity, Animals
Source:Journal of the American Chemical Society
Publisher:Amer Chemical Soc
Page Range:4181-4191
Date:1 January 2004
Official Publication:https://doi.org/10.1021/ja038774d
PubMed:View item in PubMed

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