In the classical P450 catalytic cycle (Fig. 5.2), the delivery of the first electron to the P450 heme iron reduces it from the ferric (Fem) to the ferrous (Fe11) form. The Fe11 binds dioxygen, converting to a ferrous-dioxy complex. The timely delivery of the second electron converts this species to a ferric peroxy form. Thereafter, the species is protonated to form a ferric hydroperoxy intermediate, and then further protonated to form a high valent iron-oxo complex (compound I) with release of a molecule of water through heterolytic cleavage of the dioxygen bond in the preceding intermediate. Compound I is considered to be the intermediate catalyzing the majority of P450 reactions, although the ferric hydroperoxy intermediate may also participate in some P450-dependent catalytic reactions .
Since most P450 redox systems use electrons derived from the oxidation of the coenzymes NAD(P)H, the two electrons required for P450 oxidations should be provided to the redox partner as a hydride ion from the coenzyme. However, as is clear in the cycle shown in Fig. 5.2, these electrons are then required to be delivered singly at distinct points in the catalytic cycle - either side of the binding of dioxygen to the heme iron. Thus, the role of the redox partner systems is to take two electrons (as hydride ion) from NAD(P) H and to deliver these one at a time to the P450. The first electron is donated to the Fe111 form, reducing it to Fe11.
In systems such as P450cam and the Bacillus megaterium flavocytochrome P450BM-3 (CYP102A1, see below), there is clear regulation over the transfer of electrons to the ferric heme iron mediated by the binding of substrate molecules (camphor and its analogs for P450cam, fatty acids for P450BM-3) [12, 13]. For these "well-regulated" P450s, binding of substrate in the active site induces the dissociation of a water molecule that is bound relatively weakly as the 6th coordinating ligand (trans to the thiolate) to the heme iron. This, in turn, induces an equilibrium shift of the heme iron spin-state (from low-spin S = 1/2 to high-spin S = 5/2) and a positive shift in heme iron reduction potential of the order of 130-140 mV [12, 13].
The increased potential brings the heme iron into range for efficient electron transfer from the redox partner (Pd for P450cam, a fused CPR for P450BM-3), and ensures that rapid electron transfer to the heme iron occurs only when substrate is available for oxygenation . Similar substrate-dependent heme iron dehydration and redox potential shifts are seen in other bacterial P450 systems [e.g. 15, 16], and substrate-induced heme iron spin-state shift is also observed widely in eukaryotic P450 systems [e.g. 17]. However binding of substrate to P450s is not always a prerequisite for electron transfer from the redox partner, and this brings with it the likelihood of wastage of the reducing equivalents in the non-specific reduction of bound dioxygen - so called "uncoupling" of the P450 catalytic cycle (see Fig. 5.2 and the more detailed discussion below) .
Fig. 5.2 Catalytic cycle of cytochrome P450. The resting state of a P450 is the ferric (Fem) form, usually with water present as the distal ligand to the heme iron. In the first step, the water is displaced by binding of a substrate (R-H). The first electron (e-) transfer from the redox partner reduces the complex to the ferrous (Fe11) form, which can then bind dioxygen (O2). The ferrous-oxy complex formed may also be presented as the isoelectronic ferric-superoxy form. The delivery of a second electron converts this to a ferric peroxy species, which is then protonated to the ferric hydroperoxy state, and then to compound I (presented as a ferryl-oxo porphyrin radical cation) with loss of water. Compound I oxygenates the bound substrate (to R-OH) and product dissociation leads to restoration of the ferric state and reassociation of water as the distal ligand to complete the catalytic cycle. Collapse of unstable oxy intermediates leads to uncoupling of the cycle and reformation of the ferric form of P450. The ferrous oxy complex dissociates superoxide, while the ferric hydroperoxy complex collapses with peroxide generation. The reaction can be forced in the productive direction by addition of hydrogen peroxide (or an organic peroxide) to resting substrate-bound P450 (the so-called "peroxide shunt" pathway). Collapse of compound I occurs with generation of water .
Following delivery of the first electron, dioxygen binds the ferrous iron, and then the second electron should be delivered to avoid collapse of the ferrous-oxy species back to the ferric resting state of the iron - with wasteful production of superoxide radical. Subsequent steps in the P450 cycle are considered to be relatively fast with respect to the electron transfer events, and as a result the later transient intermediates have proven difficult to characterize spectroscopically or structurally. Compelling evidence is available for the formation of the ferric hy-droperoxy intermediate, but compound I (the ferryl oxo species following immediately from ferric hydroperoxy in the catalytic cycle) has proven much more elusive [e.g. 19].
Thus, the P450 redox partners have the task of "splitting" the two electron batch derived from nicotinamide coenzyme and passing single electrons to the P450 at discrete points in the cycle. To do this, the redox partner systems usually have more than one redox active cofactor, and use the first acceptor cofactor (a flavin) to accept the hydride ion from NAD(P)H and the second donor cofactor (usually another flavin or an iron-sulfur cluster) to transport single electrons between the donor flavin and the heme iron. The composition of the major P450 redox systems is discussed below.
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