Substrate Binding Favors Enhanced NO Binding to P450_cam

Abstract

Ferric cytochrome P450cam from Pseudomonas putida (P450cam) 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 S and V 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.

Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

“…However, drug binding rates for other CYPs also have been reported in the range of 10 4 -10 5 M -1 sec -1 [48][49]. For example, binding of imidazole and chlortrimazole to Thromboxane Synthase, a 'non-classical' CYP, exhibit rates of 8.4 × 10 4 and 1.5 ×10 5 M -1 sec -1 , based on stopped-flow [49].…”

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

“…This suggests that autoxidation may be a key factor that determines the uncoupling behavior and production of toxic superoxide by cytochromes P450 in humans. The temperature dependences of autoxidation rates were analyzed according to the Eyring equation in order to derive the activation enthalpies and entropies of this reaction in the absence and in the presence of substrates (46). For CYP3A4, we obtained similar free energies of activation (Fig.…”

Mechanisms of Nitric Oxide Reactions Mediated by Biologically Relevant Metal Centers

“…Table 2 provides some examples of rate constants measured for various ferrous and ferric heme proteins and models [63,[81][82][83][84][85][86][87][88][89][90][91][92][93] and illustrates the range of k on and k off values found for ferriheme and ferroheme proteins. As noted above, the small values of k off for the latter lead to very large K NO 's, although ferrous cytochrome c (Cyt II ) is an exception.…”