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Reactions and Ligand Binding Properties of Cytochrome P450.

Ridd, Thomas Ian. (1995) Reactions and Ligand Binding Properties of Cytochrome P450. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

One of the most interesting and characteristic reactions catalysed by cytochrome P450 is the hydroxylation of alkanes and unactivated alkyl groups (O2 + 2H + RH → ROH + H2O), which is always accompanied by some degree of "uncoupling" to produce hydrogen peroxide (O2 + 2H → H2O2); added hydrogen peroxide can also be used by some cytochrome P450 enzymes for the hydroxylation of alkanes (H2O2 + RH → ROH + H2O). Major unanswered questions concerning these reactions include: the nature of the key hydroxylating intermediate and the possible role of the two conformers observed in the reduced state with ligands such as isocyanides and pyridine (corresponding to Soret bands at 440-450 and 420-430 nm). These questions were investigated by studying (i) the reactions of oxidised CYP2B4 with H2O2 and lauric acid and (ii) equilibria involving reduced CYP2B4 with a range of nitrogen bases. It was shown that purified CYP2B4 catalysed the hydroxylation of lauric acid by hydrogen peroxide producing the ω-1 and ω-2 hydroxymetabolites against a background of bleaching of the porphyrin ring with no detectable intermediate in either reaction. Iodosobenzene on the otherhand, which is known to produce the ferryl FeO3+ derivative, does not support hydroxylation or bleaching and further inhibits bleaching by added hydrogen peroxide and that the rates of both bleaching and hydroxylation increase with the concentration of H2Q2 (pK 11.7) but are essentially pH-independent. The available evidence indicates that the key intermediate in both hydroxylation and bleaching is the undissociated hydrogen peroxide (H2O2) coordinated to the Fe3+ ion. The nature of the equilibrium between conformers I and II, reported for complexes of reduced cytochrome P450, was further investigated through the use of three families of nitrogenous bases in order to test the relative importance of σ donor and π acceptor capacity and the α effect. Equilibrium was systematically shifted in favour of II by increasing the pK in all three families, indicating the predominance of σ donor over π acceptor capacity, as well as by lowering the pH. This is in agreement with the postulated equilibrium between thiol/thiolate as the proximal ligand in conformers II and I respectively. Ligands such as hydroxylamine and pyridazine, which exhibit the α effect were anomalous in showing an unexpectedly high stabilisation of conformer II, which was observed with CYP4A1 and CYP2E1 as well as CYP2B4 and provides a model for the similar anomalous behaviour of oxygen as a ligand.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Ridd, Thomas Ian.
Date : 1995
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1995.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:38
Last Modified : 06 May 2020 14:47
URI: http://epubs.surrey.ac.uk/id/eprint/856361

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