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The P450 Catalytic Cycle

 

The principle physiological rôle of the P450 superfamily of enzymes is that of a monoxygenase. The catalytic reaction can be summarised

equation1363

where RH can be one of a large number of possible substrates.

The catalytic cycle, shown in Figure gif, may be summarised as follows:

   figure1367
Figure: The catalytic cycle of cytochrome P450. The intermediate states enclosed in a dashed box have not been directly observed and are hypothetical.

  1. Substrate binding

    The binding of a substrate to a P450 causes a lowering of the redox potential by approximately 100mV [111], which makes the transfer of an electron favourable from its redox partner, NADH or NADPH. This is accompanied by a change in the spin state of the haem iron at the active site (See Subsection gif for a detailed discussion). It has also been suggested that the binding of the substrate brings about a conformational change in the enzyme which triggers an interaction with the redox component [112].

  2. The first reduction

    The next stage in the cycle is the reduction of the tex2html_wrap_inline3186 ion by an electron transfered from NAD(P)H via an electron transfer chain.

  3. Oxygen binding

    An tex2html_wrap_inline2954 molecule binds rapidly to the tex2html_wrap_inline3190 ion forming tex2html_wrap_inline3192 . There is evidence to suggest that this complex then undergoes a slow conversion to a more stable complex tex2html_wrap_inline3194 [113].

  4. Second reduction

    A second reduction is required by the stoichiometry of the reaction. This has been determined to be the rate-determining step of the reaction [114]. A comparison between the bond energies of tex2html_wrap_inline2954 , tex2html_wrap_inline3198 , and tex2html_wrap_inline3200 suggest that the tex2html_wrap_inline3202 complex is the most favourable starting point for the next stage of the reaction to occur [77]. However, evidence from resonance Raman spectroscopy indicates the presence of a superoxide ( tex2html_wrap_inline3198 ) complex [115].

  5. tex2html_wrap_inline2954 cleavage

    The tex2html_wrap_inline3200 reacts with two protons from the surrounding solvent, breaking the O-O bond, forming water and leaving an tex2html_wrap_inline3210 complex.

  6. Product formation

    The Fe-ligated O atom is transferred to the substrate forming an hydroxylated form of the substrate.

  7. Product release

    The product is released from the active site of the enzyme which returns to its initial state.

The structures of the transitional states following processes (4) and (5) have never been directly observed and are hypotheses based on analogy with other hemoproteins and often conflicting experimental evidence.

In the investigations described in this chapter we focus on the system during the first stage of the reaction process, between (1) and (2) in Figure gif.


next up previous contents
Next: Physical Properties of P450 Up: Cytochromes P450 Previous: History and Background

Matthew Segall
Wed Sep 24 12:24:18 BST 1997