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

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

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

  
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 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 ion by an electron transfered from NAD(P)H via an electron transfer chain.

3. Oxygen binding

   An molecule binds rapidly to the ion forming . There is evidence to suggest that this complex then undergoes a slow conversion to a more stable complex [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 , , and suggest that the 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 ( ) complex [115].

5. cleavage

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