Cytochrome P450 catalytic cycle (How Oxidative Systems Metabolize Substrates) (Human Drug Metabolism)

Having established the phenomenal multiplicity and flexibility of these enzymes, it should be a relief to learn that all these enzymes essentially function in the same way, although again we do not fully understand the process yet. CYPs can carry out reductions (see later on) and these occur after substrate binding and before oxygen binding. However, their main function is to insert an oxygen molecule into a usually stable and hydrophobic compound.However, it is important to understand that there are only five main features of the process whereby the following equation is carried out:

Hydrocarbon (-RH) + O2 + 2 electrons + 2 H + ions gives: alcohol (-ROH) + H2O

1.    Substrate binding (reduction may happen after this stage).

2.    Oxygen binding.

3.    Oxygen scission (splitting).

4.    Insertion of oxygen into substrate.

5.    Release of product.

Substrate binding

The first step, as covered in the previous section, is the binding and orientation of the molecule. This must happen in such a way that the most vulnerable part of the agent must be presented to the active site of the enzyme, the iron, so the molecule can be processed with the minimum of energy expenditure and the maximum speed. The iron is usually (but not always) in the ferric form when the substrate is first bound:


Once the substrate has been bound, the next stage is to receive the first of two electrons from the REDOX partners, so reducing the iron:


Oxygen binding

The next stage involves the iron/substrate complex binding molecular oxygen sourced from the lungs. This process runs faster than the substrate binding to the iron, as there is much more oxygen present in the cell than substrate.


You will note that oxygen does not just exist as one atom. It is much more stable when it is found in a molecule of two oxygen atoms, O2. Indeed, oxygen is almost never found in nature as a single atom as its outer electron orbitals only have six instead of the much more stable eight electrons. To attain stability, two oxygen molecules will normally covalently bond so sharing four electrons, so this gives the same effect as having the stable eight electrons. So therefore, to split an oxygen molecule requires energy, but this is like trying to separate two powerful electromagnets – the oxygen will tend to ‘ snap back’ immediately to reform O2 as soon as it is separated. Two problems thus arise: first, how to apply reducing power to split the oxygen and second, how to prevent the oxygen reforming immediately and keeping the single oxygen atom separate long enough for it to react with the vulnerable hydrocarbon substrate.

Oxygen scission (splitting)

To split the oxygen molecule into two atoms firstly requires a slow rearrangement of the Fe2+ O2 complex to form


The next stage is the key to whether the substrate will be oxidized or not. This is the rate-limiting step of the cycle. A second electron from supplied by the REDOX partners feeds into the complex and forms




As stated earlier, it has been suggested that of the two REDOX partners, cytochrome b5 may supply this second electron more quickly than NADPH reductase. As this stage of the process is so rapid it is not feasible to detect experimentally, so the most likely pathway has been worked out which corresponds to what is possible and what happens in terms of the products that we can actually measure. Certainly an oxygen atom with two spare electrons is a very attractive prospect to two hydrogen atoms and water is formed, leaving a single oxygen atom bound to the iron of the enzyme. This solves the two problems described above; the oxygen molecule has been split, but it cannot just ‘snap back’ to form an oxygen molecule again, as water is stable and takes an oxygen molecule away from the enzyme active site.

Insertion of oxygen into substrate

The remaining oxygen is temporarily bound to the iron in a complex, which is sometimes termed a ‘perferryl’ complex (below).


It has been suggested that the perferryl complex may operate in low or high spin states and also that oxygen is bound in different ways to the iron. FeO2+ (peroxo-iron) and even a peroxide (FeOOH2+) have both been suggested to take part in some CYP reactions such as oestrogen aromatizations. The perferryl is thought to be the main method of oxygen binding to the CYP and it is exceedingly reactive, as it activates the substrate by either removing hydrogen (hydrogen abstraction) or an electron (e.g. from nitrogen atoms) from part of the substrate molecule. These steps are not necessarily in that order and multiple electron or abstractions can take place. It is apparent that the hydrogen abstraction part of the process takes longer than the subsequent processes and is thought to be the rate-limiting step in the oxidation process. The hydrogen to be removed will be closest to the carbon to be oxidized. The abstracted hydrogen is then bound to the perferryl complex. This leaves the carbon with a spare electron, which makes it a reactive radical, as seen below. The substrate, i.e. the carbon atom, has been activated which makes sense as it is now much more likely to react with the hydroxyl group.


The final stage is the reaction between the newly created hydroxyl group and the carbon radical, yielding the alcohol, as seen below. The entry of the oxygen atom into the substrate is sometimes called the ‘oxygen rebound’ reaction.


Release of product

The whole CYP catalytic process could be described as complex yet dramatic. Although a ‘pump’ analogy has been used previously in this topic, in some ways, CYPs could also be likened to the cycling of an automatic weapon, with the ‘load, fire, extract, eject, reload’ stages analogous to CYP substrate conversion to product. This analogy is not perfect, as the coupled CYP provides all the energy to sustain the process rather than the ‘substrate’ of a machine gun, but it conveys something of the rapidity and violence of the process. Once the substrate has been converted to a metabolite, it has changed both structurally and physicochemically to the point that it can no longer bind to the active site of the CYP. The metabolite is thus released and the CYP isoform is now ready for binding of another substrate molecule. It is important to break down the function of CYPs to separate stages, so it can be seen how they operate and overcome the inherent problems in their function. However, students often find the catalytic cycle rather daunting to learn and can be intimidated by it. It is much easier to learn if you try to understand the various stages, and use the logic of the enzyme’s function to follow how it overcomes the stability of substrate and oxygen by using electrons it receives from the adjacent reductase systems to make the product. A simplified cycle is shown on Figure 3.5. As more research is carried out, fine details may change in the cycle, but the main features, the substrate binding, option for reduction, oxygen binding and activation, perferryl complex formation, abstractions of hydrogen or electrons and finally substrate release are well established.

Simplified scheme of cytochrome P450 oxidation

Figure 3.5 Simplified scheme of cytochrome P450 oxidation


As mentioned earlier, CYPs probably evolved to reduce chemical agents before oxidation could occur and once an agent is bound to the haem iron, there is an opportunity for a reduction reaction to occur prior to the oxygen binding stage. This is because a REDOX partner, usually NADPH reductase, supplies an electron which can be used to reduce the substrate. There are several enzyme systems that can effect reductions, such as NADPH reductases themselves, which are found in virtually all tissues and are relevant in aromatic amine-mediated carcinogenicity.CYPs are just one of many systems which can reduce xenobiotics and interestingly, there are examples where drugs undergo reductions and oxidations sometimes by the same CYPs. This is thought to occur with the muscle relaxant eperisone and human CYPs are capable of reducing the antibiotic chloramphenicol also.

Next post:

Previous post: