The formal stoichiometry of a P450 reaction would be:

1 RH (organic substrate) + 1 NADPH + 1 O₂ ⇒ 1 ROH (product) + 1 NADP⁺ + 1 H₂O

This is, however, rarely the case. A few P450s are “well coupled”, for instance bacterial P450cam (CYP101) hydroxylates camphor with this stoichiometry (with NADH instead of NADPH). In most cases, the reaction is somewhat “uncoupled” with more NADPH and more molecular oxygen consumed and some side products, such as hydrogen peroxide or superoxide produced. This is a result of the complex reaction cycle described below.

The house fly CYP6A1 is to date the only arthropod P450 for which a stoichiometry has been measured (Murataliev et al., 2008). The numbers for heptachlor epoxidation show how important “uncoupling” can be for a promiscuous enzyme such as CYP6A1. (Note that superoxide production was not measured directly and would be accounted for in the water and hydrogen peroxide values).

1 heptachlor + 11.2 NADPH + 13 O₂ ⇒ 1 ROH (product) + 11.2 NADP⁺ + 1.5 H₂O + 9.8 H₂O₂

Substrate-dependent uncoupling of the P450 enzymes that protect arthropods from their chemical environment is therefore a corollary of the detoxification process, and the toxicological as well as signaling effects of this oxidative stress generated by P450-dependent metabolism deserves further study. This would be particularly important in the case of P450-mediated pesticide resistance, when arthropods depend on increased metabolism of a toxic xenobiotic for survival. Oxidative stress caused by uncoupling of the constitutively overexpressed P450(s) may constitute a fitness cost of resistance.

NADPH is an essential cofactor of arthropod P450 reactions, as the stoichiometry (above) indicates. The reducing equivalents from NADPH are transferred to P450 enzymes from two types of redox partners: (a) for microsomal P450s, from NADPH cytochrome P450 reductase , with in some cases a contribution from cytochrome b5, and (b) for mitochondrial P450s, from insect orthologs of adrenodoxin and adrenodoxin reductase.

Little work on insect P450 has focused on the catalytic cycle, so the mechanism derived from our understanding of the bacterial and mammalian P450 enzymes is briefly summarized.

The oxidized P450 is a mixture of two forms: a low spin Fe(III) form with water as the sixth coordinated ligand on the opposide side of the Cys thiolate ligand, and a high spin Fe(III) pentacoordinated form. Substrate binding can displace water from the sixth liganding position, leading to a blue shift from low spin (~417 nm peak in the absolute spectrum) to high spin (~390 nm). This shift can be observed (Type I difference spectrum) and is accompanied by a decrease in the redox potential of P450.

The P450-substrate complex receives a first electron from a redox partner (P450 reductase or adrenodoxin), and ferrous P450 Fe(II) then binds O₂. At this step CO can compete with O₂ for binding to P450, its binding leads to a stable complex, with absorption maximum at 450 nm, that is catalytically inactive. CO can be displaced by light irradiation at 450 nm.

The P450-O₂-substrate complex then accepts a second electron (from P450 reductase or in some cases cytochrome b5, or from adrenodoxin) to form a ferric peroxide anion. After protonation, a ferric hydroperoxo complex then leads to the activated oxygen form(s) of the enzyme. Although the formal reaction is the insertion of one atom of oxygen into the substrate (hence the term “monooxygenase”), the other atom being reduced to water (hence the term “mixed-function oxidase”), the precise nature of the oxidizing species, compound I (by analogy to compound I of peroxidases) has long remained elusive. This intermediate is an Fe(IV) oxo species with a delocalized oxidizing equivalent (Rittle and Green, 2010). Hydroxylation of an unactivated C-H bond therefore follows a “rebound” mechanism, where hydrogen is abstracted from the substrate by compound I, forming an Fe(IV) hydroxide that then recombines quickly with the substrate radical: