In this review, attempts have been made to (a) bring together published information consistent with the two-site behavior of P450 active site in binding of substrates, products and inhibitors. The two sites are the substrate binding site the specific protein residues (Site I) and the L6 position of the iron (Site II); (b) to present more recent results from the author’s laboratory obtained with a fully coupled system (P450cam) on substrate and product interactions which are also consistent with two site behavior of the active site and inconsistent with the single-site model; and (c) to discuss the implications of the substrate and product interactions with the sub-sites for coupling/uncoupling; (d) present more recent kinetic data indicating that Type II binding which involves binding to the buried Site II, is a two step reaction consistent with the differences in the accessibilities of the two sub-sites.
History of assigning a function for P450 as hydroxylase: I have had the opportunity to personally experience the inception of the functional aspects of the P450 field, as an independent investigator, while I was associated with Dr. Otto Rosenthal . Therefore, I would like to share some of my early experiences with those who visit my home page.
Ryan and Engel had demonstrated (J. B. C, 225, 103-114, 1957) that carbon monoxide inhibits steroid C-21 hydroxylation reaction in bovine adrenocortical microsomes, and that this inhibition is removed by visible light, indicating participation of a CO-sensitive pigment in the reaction. However the presence of such a pigment could not be demonstrated because the microsomes were too turbid for spectrophotometry with the then conventional instruments, and their attempts to solubalize them had inactivated the enzyme. In early 60s, extracts of the microsomes which were highly active for steroid C21 hydroxylation and suitable for spectrophotometry were prepared, and the presence of the CO-sensitive pigment (cytochrome P450) in these extracts was demonstrated . In this preparation, the electron donor preference for reduction of the pigment was the same as that for the overall hydroxylation reaction. In addition, the natural substrate 17-OH progesterone was capable of binding to the P450 as indicated by the spectral shifts in the cytochrome . In the presence of the specific electron donor and O2, the spectral change produced by less than saturating concentration of the substrate largely disappeared as the substrate was converted to the product . In addition, CO the inhibitor of the overall reaction inhibited the disappearance. When P450 was converted to its inactive derivative P420, the hydroxylation activity was lost and the substrate failed to elicit the characteristic spectral changes. These observations were as would be expected if the spectral change represented the usual enzyme-substrate complex. Therefore, it was considered very promising that the P450 in this preparation was the CO sensitive pigment participating in the hydroxylation reaction, and that the pigment displaying the substrate-induced spectral changes was P450 itself. This sequence of events was the basis for all our subsequent work in our laboratory including the application of the photochemical action spectroscopy by Estabrook, Cooper and Rosenthal ( Biochem. Z. 338, 741-755, 1963), as also indicated in Ref: . To my knowledge, all of these findings which were available in our laboratory around 1961-62 were the starting point for the functional aspects of the P450 field.
Since then numerous P450s have been discovered. At present there are 267 families with more than 5000 genes (http://drnelson.utmem.edu/cytochrome P450.html). These ubiquitous enzymes have many applications. They catalyze many physiologically, pharmacologically and toxicologically important hydroxylation reactions, in the presence of specific electron transport system and molecular oxygen. In a different application, the microbial enzymes P450cam can be genetically engineered to detoxify environmental toxins. Since the P450 enzymes can catalyze hydroxylations of unactivated centers in hydrocarbons, these enzymes can be used for synthesizing compounds that are not accessible by chemical means. It is important to understand the determinants of catalytic efficiencies of these enzymes for all applications. When a P450 system is operating at full catalytic efficiency, the reducing equivalents and oxygen consumed are totally coupled to substrate hydroxylation. When the system is uncoupled, O2 and reducing equivalents consumed are deviated from normal pathway of substrate hydroxylation, producing side products of O2 reduction H2O , O2-, & H2O2 (Nordbloom & Coon, ABB. 1977, 180, 343). The latter two can be converted to toxic .OH radicals, in the presence of reduced transition metals such as iron in P450 itself. The .OH radicals can cause unpredictable toxic effects on the cell. Majority of drugs used today undergo partially uncoupled metabolism by P450 enzymes, which could significantly contribute to drug toxicity. Understanding molecular basis for coupling/uncoupling is of utmost importance for all applications of cytochrome P450, of particular interest, is for designing safer drugs. Current screening procedures for drug candidate selection may detect the problem, but they cannot offer any reasonable insights for finding a solution to the problem. The solution requires thorough understanding of molecular mechanisms of coupling/uncoupling. A P450 system can become uncoupled in more than one way, for example improper orientation of a substrate, untimely water access to the O2 binding site. Therefore it is difficult to be certain about the observed cause and effect relationships. My laboratory is currently interested in understanding how a fully coupled system can avoid all different ways of uncoupling. The approach is based on the differential behavior of the sub-sites of the P450 active site in binding of substrates and products. This is explained in  and summarized in the ensuing paragraphs.
The P450 enzymes share a common reaction cycle in regard to spin and coordination states of the ferric enzyme and their abilities to undergo reduction. Although there are several substrate-free high spin P450s (Black & Coon, Rev. Adv. Enzymol, 1987, 60, 35) majority of the substrate-free P450s are low spin and six coordinated with a water as L6 ligand, first observed with P450cam (Poulos et al, Biochem, 1986, 25, 5314). It is unlikely that the L6 ligated enzyme can undergo reduction therefore, it has been referred to as resting state. In the P450 catalytic cycle, there are two one electron transfer steps. The substrate binds to the low spin converting it to the five coordinated high spin reducible (functional) complex (ESHS), which undergoes first electron reduction, followed by O2 binding to form the ternary complex Sub-Fe++O2, with substrate and O2 bound to their respective sites. This is an important intermediate in the formation of the hydroxylating species. Then the P450 active site must be considered as having two different sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the more accessible substrate binding site the specific residues of the protein moiety (will be referred to as Site I) and the L6 position of the buried iron to which O2 binds upon reduction (will be referred to as Site II). In the ferric P450, certain amines and –OH compounds such as products of P450-catalyzed reactions can serve as excellent Site II ligands and they form low spin six coordinated inhibited complexes ((Dawson et al., J. B. C., 1982, 257, 3606; White & Coon, J. B. C., 1982, 257, 3073). The X’ray and spectroscopic data on the bacterial enzyme P450cam which has served as a model system, have shown that (a) the substrate camphor binds to Site I and releases the water ligated at Site II forming five coordinated functional complex (Poulos et. al, J. Mol. Biol, 1987, 195, 687); and (b) the product 5-exo-OH camphor through its –OH group binds at L6 forming Fe-O bond with appreciable strength resulting in six coordinated inhibited complex (Li et al, J.A.C.S, 1995, 117, 6297& ). Therefore substrate and product interactions with the two sub-sites can determine when to turn on and turn off the enzyme during catalysis. The presence of the substrate or the product has been shown to protect preformed oxy complex (Fe++O2) of P450cam (Lipscomb et al., J. B. C., 1976, 251, 1116) possibly by blocking water access to the oxygen bound site. It is well known that aqueous environment can destabilize oxy heme complexes. Therefore fine tuning of the on and off events in such a way as to sequester the oxy, peroxo, peroxy and ferryl intermediates of the catalytic cycle from bulk water could avoid uncoupling (Poulos & Raag, FASEB J. 1992, 6, 674). Since substrate binding to Site I can release the L6 water from Site II despite the inability of the substrate to bind to this site, suggests inhibitory allosteric effect of Site I binding on Site II ligation. That Site II ligation can inhibit Site I binding is suggested by comparison of the static and kinetic KDS’ for certain substrates and products . Therefore interactions between the two sub-sites can contribute to fine tuning of the on and off events.
|Figure. 1. Catalytic cycle showing the role of ESP in
replenishing ESHS bypassing ELS which has L and
other waters. Proposed to be fully coupled cycle.
The two-site behavior of the P450 active site is also reflected in binding of camphor (S) to P450cam (E) in the presence of the product (P) . The data could not be fit to the single-site competition model in which the S and P strictly compete for the same site. The data gave excellent fit to the two-site model involving the ternary complex ESP. The S binds with much higher affinity to EP than P binds to ES. Therefore when the S binds to EP to form ESP, the P dissociates resulting in the high spin complex ESHS. Thus the relative values of the parameters of the equilibria involved are compatible for ESP to serve as an important intermediate for replenishing the first electron acceptor ESHS bypassing the resting state (ELS) as shown in the Figure 1. Bypassing the resting state ELS which has L6 water will also bypass structures which must allow water access to restore the resting state. This is important for sequestering the oxygen activating machinery from water (Poulos, Drug Met. & Disposition, 2005, 33, 10-18).
Why do we have to take into Account Differential Behavior of the Sub-sites of the Active Site for Understanding P450 Enzymes?
As indicated earlier, P450 catalytic cycle involves two one electron transfer steps. We have observed different classes of P 450 behavior in regard to the first electron transfer (reviewed in Ref:  ). These are (i), Low spin six-coordinated not reducible P450s which are readily converted to the five-coordinated reducible high-spin state upon substrate binding, and express appreciable hydroxylation activities; (ii), Substrate-free high spin P450s, which undergo reduction even in the absence of substrates, and are functionally fully competent despite the absence of the low spin state; and (iii) Low spin P450s, which express appreciable hydroxylation activities in the absence of observable substrate-induced low- to high-spin transition, as indicated by the absence of Type I spectral change, observable in the timescale of the routinely used traditional static spectroscopy. Our rational approaches based on the single-site view of the active site have led us into concluding that different P450s are different, and that some low spin P450s need to become high spin for activity, some are already high spin and they do not need low spin for activity, and some others function with only low spin because substrate-induced low spin to high spin was not observable by the static spectroscopy.
On the basis of the two-site behavior of the P450 (E) active site, these differences between different P450s are reasonable within the frame work of the same model, the two-site model for substrate (S) and product (P) binding to E involving ESP. This is explained as follows: As indicated earlier, the parameters of the equilibria of the two-site model are compatible for ESP to serve as an important intermediate for replenishing the ESHS the starter of the catalytic cycle, bypassing ELS , indicating that the resting state is not an intermediate in the cycle. This then is consistent with the existence of substrate-free high spin enzymes (EHS), which are fully competent, despite the absence of the low spin species (ELS). This indicates that same catalytic cycle (Fig. 1) is applicable to both low spin and high spin enzymes, except that the regulatory step reflecting the conversion of the not reducible (ELS) to the reducible high spin state (ESHS) in the first cycle does not exist. In regard to the ES complexes which are observable as low spin in their static spectra, the transient high spin intermediates observable in the time-scale (sub-millisecond) of the T-jump relaxation technique , suggest an interesting possibility that the substrate-bound low spin complexes may work through the transient high spin intermediates. This then would be in-line with the requirement for the high spin for reduction, and the requirement for the five-coordinated state with L6 free for O2 binding to the reduced enzyme.
The overriding hypothesis  is as follows: Strikingly different properties of the differently accessible sub-sites, and inhibitory allosteric interactions between the two sites, determine the thermodynamic and kinetic parameters of substrate and product interactions with the two sub-sites, which could regulate the five coordinated high spin functional and the six coordinated low spin inhibited states during catalysis. This in turn, could determine the catalytic efficiency i.e. degree of coupling/uncoupling as discussed earlier and in greater detail in Ref: . My laboratory at present is interested in further substantiating this hypothesis.
Summary: There is considerable published information indicating that P450 enzymes cannot be treated as simple single site enzymes in binding of substrates and product and also inhibitors . Especially because the Site I complexes are functionally different from the Site II complexes, the two sub-sites cannot be considered as parts of a site functioning as single site. As indicated earlier, I have been in the P450 field since the inception of its function as hydroxylase. A new comer into the field will find it difficult because there are reviews and other write ups assuming that P450 is a single site enzyme. This single-site view has not given us any insights into how a fully coupled P450 can avoid all different ways of uncoupling. When an important aspect of a research field becomes controversial, it is not uncommon to take a look at the experimental bases for the models. In this regard the selected publications [23, 24] may be helpful to a new comer.