Cytochromes P (CYPs) are proteins of the superfamily containing heme as a cofactor and, . This pathway entails oxidation of the ferric-substrate complex with oxygen-atom donors such as peroxides and hypochlorites. Cytochrome P enzymes are present in most tissues of the body, and play important roles in. Cytochrome P enzymes are responsible for most phase I reactions. · Phase II metabolism takes place if phase I is insufficient to clear a compound from. Cytochromes P (P) are membrane-bound enzymes that catalyze the monooxygenation of a diverse array of xenobiotic and endogenous.
P450-complex enzymes Cytochrome
Human Ps have been shown to also catalyze steps in morphine synthesis supplemental Fig. Most of these reactions are internal couplings, in which rings are in juxtaposition to fuse if a radical pathway is initiated. The coupling of two flaviolin molecules together has been observed in a Streptomyces coelicolor CYPA2 reaction, in which the two molecules are both bound in the active site A dimer of capsaicin is formed during its oxidation by human liver microsomes although which P is involved remains unknown The general reaction supports a view of an aromatic radical pathway but is rare among oxidations of unlinked substrates.
Recently, the environmental contaminant BDE, a polybrominated bis -phenyl ether flame retardant, was reported to be converted to a brominated p -dioxin molecule by the introduction of a second ether oxygen supplemental Fig. A highly unusual reaction is involved in the synthesis of a phytotoxin, thaxtomin, in Streptomyces turgidiscabies Fig.
This is not a physiological reaction but nevertheless demonstrates the versatility of Ps this is not an oxidation per se but is included here in the context of mechanistic similarity. Multiple turnovers were observed, and the stereochemistry was distinct from that seen in reactions with only free heme.
The activation process includes both a spin-state change in the iron and a constriction in the size of the active site. Watanabe and co-workers 46 were not able to oxidize methane or ethane in their system, but Reetz and co-workers 45 were able to achieve multiple turnovers of methane, an unusual feat.
Although the vast majority of P reactions are oxidations, reductions are also known. Most are observed more readily under anaerobic or hypobaric conditions. For rather unclear reasons, the substrates for such reactions accept electrons from ferrous P at a rate that is competitive with the rates of binding and reduction of oxygen.
These reactions can occur in cells with low oxygen tension, e. The electrons must be transferred one at a time. The nature of the 1-electron reduced intermediates and, in some ways, the fate of an oxygen in a substrate remain unclear. One unusual case is the conversion of benzo[ a ]pyrene epoxides to the polycyclic hydrocarbon itself, i.
At least three non-redox P reactions have been reported, including a phospholipase D-type hydrolysis by several mammalian Ps 51 , pyrophosphatase hydrolytic activity of S. Those reactions are likely to involve elements of acid-base chemistry, but the details are unknown. In the case of CYPA1, an alternative active site for the hydrolytic reaction has been identified the P also catalyzes oxidative reactions.
Typical prokaryotic Class I systems have similar flavoprotein reductase and ferredoxin FDx partners Diversity of P redox systems and P fusion proteins. A selection of distinct types of P enzymes and where relevant their redox partner systems is shown.
The sizes of the boxes are indicative of the lengths of the protein modules. Bound prosthetic groups are indicated in the color-coded domains. This protein is depicted with FAD bound in its N-terminal domain, but there is no report to date of characterization of this protein. F , Mimivirus CYPA1, with a P fused to a C-terminal domain of uncertain function but containing several potential sites for post-translational modification.
Most bacterial systems use a similar redox apparatus L , variation on system K, in which a flavodoxin replaces the iron-sulfur protein. The discovery of a high-activity fatty acid hydroxylase in Bacillus megaterium led Narhi and Fulco 65 to identify the first P linked to its redox partner.
Both domains lack the membrane anchor regions typical of their eukaryotic relatives, and P BM-3 was the first example of a prokaryotic CPR P BM-3 is a dimeric enzyme, and intermonomeric electron transfer occurs in this system, as also seen for the structurally related eukaryotic nitric-oxide synthases 70 — The physiological function of P BM-3 still remains unclear, although a role in bacterial quorum sensing mediated by oxidative inactivation of acylhomoserine lactones has been proposed Similar types of PCPR fusion enzymes have been found in lower eukaryotes, most notably the Fusarium oxysporum membrane-associated fatty acid hydroxylase P foxy CYP Several years elapsed between the discovery of P BM-3 and the characterization of the next type of Predox partner fusion enzyme.
Genome sequence data led to the identification of a small number of bacterial Ps fused to partners resembling phthalate dioxygenase reductases In the case of CYPB1, the enzyme was shown to be catalytically active in the oxidation of thiocarbamate herbicides, consistent with the sequence similarity between its P domain and the herbicide-degrading Class I P CYPA1 from Rhodococcus sp. The CYPB-type model has recently been mimicked in artificial P fusion enzymes in efforts to enhance eukaryotic P activities and produce metabolites of interest e.
XplA receives electrons from an NADPH-dependent flavoprotein reductase XplB and catalyzes the reductive degradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine, producing nitrite as a final product A flavodoxin-like module in CPR is ultimately responsible for electron delivery to mammalian microsomal Ps, and flavodoxins were also shown to support electron transfer to the bacterial Ps involved in biotin synthesis and cineole catabolism, replacing ferredoxins in Class I-like systems 62 , In doing so, these Ps either use an alternative route to achieve substrate oxidation or exploit the P scaffold for unconventional catalytic functions.
The peroxide is rarely efficient and is often highly destructive in oxidizing the protein and the heme prosthetic group non-physiological high-valent iodine compounds, e. However, this class of Ps uses this mechanism and avoids excessive oxidative damage during the process.
A more recently discovered member of the CYP P family is the OleT P from Jeotgalicoccus ATCC , which uses the peroxygenase route to decarboxylate a range of long chain fatty acids to form the n -1 alkenes. OleT thus has potential applications for biofuel and fine chemical synthesis. There are separate cytosolic and mitochondrial isoforms of P nor found in F. Selected Ps are known to catalyze isomerization reactions.
TXA 2 causes vasoconstriction and induces platelet aggregation. CYP8A1 prostacyclin synthase also isomerizes PGH 2 , in this case, generating prostacyclin, which is vasodilatory and inhibits platelet aggregation These reactions thus have competing regulatory functions.
In both cases, the mechanism is proposed to involve homolytic cleavage of the PG endoperoxide, with a ferryl P iron-bonded to one or the other of the oxygen atoms depending on whether CYP5A1 or CYP58A1 is the catalyst and the particular binding mode of the substrate in the P active site i. Radical migration to a substrate carbon and electron transfer to the iron with carbocation formation on intermediates precede their final rearrangement to form either TXA 2 or prostacyclin Plant allene oxide synthases the CYP74A family catalyze dehydration of fatty acid hydroperoxides to form the respective allene oxides 93 , which are reactive epoxides that are further transformed into the plant hormone jasmonic acid.
Jasmonic acid and its metabolites are crucial in plant growth and development and defense. Genome sequencing projects have led to the identification of a number of novel P enzymes covalently attached to protein modules that are not obvious NAD P H-dependent redox partners or domains thereof. Many of these P fusions likely catalyze consecutive reactions or otherwise interact productively with the Ps to perform important physiological functions.
Characterization of this PpoA enzyme demonstrated that the peroxidase domain catalyzed oxidation of linoleic acid to form 8 R -hydroperoxyoctadecadienoic acid, with this product then being isomerized by the P domain to form 5,8-dihydroxyoctadecadienoic acid.
The simplest system that supports NADPH-dependent oxygenation reactions includes three major components: However, as membrane-bound proteins, their motion is restricted to the confines of a lipid bilayer, usually that of the endoplasmic reticulum, which localizes and orients CPR and P in a manner conducive to a functional interaction.
Other proteins are also known to contribute to monooxygenase function of P, the most notable being cytochrome b 5. This has been documented with the heme enzyme, cytochrome b 5 , which can serve as an allosteric effector without supplying electrons for Pdependent substrate metabolism Yamazaki et al. An interesting feature of the P system proteins is that the components are not present in equimolar concentrations.
In addition to supplying electrons for Pmediated metabolism, CPR also functions as a redox partner in reactions catalyzed by heme oxygenase, fatty acid desaturase and, in some cases, cytochrome b 5.
Despite its contribution to so many different reactions, CPR concentrations are generally limiting. Early estimates from untreated rat liver microsomes reported about a fold excess of P over that of CPR Estabrook et al. When CPR was measured immunochemically, the P CPR ratio was about 5: This difference in the relative concentrations of proteins that are known to form a 1: Those Ps outside the cluster received electrons at a much slower rate.
The relative ratios of P and CPR are subject to alterations caused by inducing agents: CPR ratios of about Cytochrome b 5 levels are generally in the same range as those of Ps in untreated rats Estabrook et al.
Heme oxygenase-1 HO-1 levels also are quite variable depending on induction status. CPR ratio of about 0. CPR ratio to 3: With the molar levels of CPR always being limiting when compared to the sum of all proteins that require CPR as their electron donor, changes in the ratio of CPR to individual electron acceptors would be expected to have a profound influence on drug disposition, heme metabolism, and other CPR-mediated functions such as fatty acid metabolism.
The proteins in the endoplasmic reticulum are concentrated at a relatively high protein density lipid: This condition raises several questions related to the function of the P systems as well for the behavior of other ER-resident proteins: This review will summarize studies that were designed to address these questions, focusing on the potential for different P proteins to form physical complexes within the endoplasmic reticulum, and their potential to influence P function.
One possible explanation for these findings was that complex formation among different forms of P was influencing metabolism by the individual enzymes. Because we are primarily concerned with the manner by which PP interactions influence the metabolism, disposition, and elimination of compounds, this review begins with a summary of the studies that have examined the effects of these interactions on P activities. There are several mechanisms by which one P enzyme can affect the behavior of another form.
According to this model, we are assuming that the substrate concentration is saturating for both P enzymes, which allows us to simplify the model to focus on the protein-protein interactions. Although the substrate concentration is saturated for both Ps, each P would have its own characteristic rate of formation of a particular product. A P that binds tightly to CPR and effectively converts substrate to product would be a major contributor to the overall reaction rate.
In this model, each of the P enzymes has its own inherent ability to associate with CPR defined by the Michaelis constants K D a and K D b as well as their rates of substrate turnover defined by the rate constants k a and k b. Neither the K D nor the rate constant for either P are affected by the presence of the second P enzyme; however, at subsaturating CPR levels both Ps will compete for the available CPR with overall substrate turnover being dependent on these constants.
The species labeled in blue are capable of converting substrate to product. Now, if the substrate is selective for P a , then the substrate will be effectively metabolized in the mixed reconstituted system; however, if that substrate is not effectively metabolized by P a , then substrate turnover would be lower.
That is, a P with a smaller K D for CPR would be able to more effectively compete for the available CPR, and consequently provide a greater contribution to total product formation. In the second potential mechanism, two P enzymes can form a physical complex.
This complex can lead to a conformational change that alters the relative abilities of the Ps to bind to CPR. As shown in Model 2 , there are several species capable of forming product. There are also at least two ternary complexes with P a and P b forming a heteromeric complex, and CPR binding to one or the other P moiety of the complex shown in brown. Finally, there is the potential for the formation of a quaternary complex, with CPR binding to both P moieties of the heteromeric complex shown in green.
This would be expected to occur either in the event of a strong cooperative effect, or at saturating CPR levels. Although the P complexes are described as dimers in this model, we cannot exclude the potential for the Ps exist in the membrane as higher order complexes. In this model, P a and P b are capable of forming a physical complex that affects the ability of CPR to bind. P a and P b monomers each have their characteristic abilities to bind and metabolize substrate as described in Model 1.
According to this model, the rate constants for substrate turnover are not affected. At subsaturating CPR concentrations the reaction rate can either be stimulated or inhibited by altering the fraction of CPR bound to a particular P enzyme. The latter complex would only be expected to accumulate at saturating CPR levels or in the event of a significant cooperative effect. If the K D ab is small, there will be a greater tendency for the P enzymes to exist in a complex, which will decrease the amount of the monomeric Ps.
If the substrate used is a P a selective substrate, then the overall reaction rate would be higher than that expected based on the single P enzyme. On the other hand, if the substrate used is P b selective, an inhibition would be observed in the mixed reconstituted system. Therefore, in a Model 2 interaction at saturating CPR, the overall rate would be expected to be additive.
In these cases, the rate constants for substrate turnover will be affected. As an example, let us begin with a mixed reconstituted system described in Model 3. However, the rate of substrate turnover is affected.
This could be seen as an increase in electron transfer or any other catalytic step. If the reaction rate for the complex is faster than that of the uncomplexed enzyme, stimulation in the metabolic rate would be observed.
For example, if k d is larger than k b , substrate turnover from the mixed system would exceed the sum of the CPR-P a plus CPR-P b binary systems.
In contrast, if the k d is smaller than k b , an inhibitory response would be observed. This response, whether synergistic or inhibitory, will be observed both at subsaturating and saturating CPR, in contrast to the response described in Model 2 for alterations in CPR binding affinity.
This model is similar to Model 2 in that P a and P b can form a physical complex. This would cause either an inhibiton or synergistic stimulation of substrate turnover that would be observed at both subsaturating and saturating CPR concentrations.
In order to elucidate the functional consequences of PP interactions, many studies have been conducted in which substrate metabolism by individual Ps and mixtures of Ps were compared. As will be described, studies examining the influence of one P on metabolism by another P have produced effects consistent with each of the interaction models described above. Kinetic analysis of the rates of metabolism by reconstituted systems varying the relative amounts of the purified enzymes was shown to be consistent with simple competition for CPR.
Thus, this study examined PP interactions in natural membranes, and the results were not influenced by potential artifacts resulting from incorporation of proteins into lipid-reconstituted systems. The study also used substrates that were specific for each enzyme and did not inhibit the other enzyme, so effects on metabolism by the Ps could be compared with and without substrate turnover by the alternate enzyme.
The inhibition could be alleviated by increasing the relative levels of CPR. Significantly more inhibition was observed when the alternate enzyme was also involved in substrate metabolism indicating either that Ps bind CPR with greater affinity during metabolism or that the supply of electrons from CPR was limited when both enzymes were active.
Given these data, the study concluded that the inhibition observed in the ternary system containing both Ps and CPR could be attributed to a simple competition of the Ps for a limiting supply of CPR. A much more intriguing effect of PP interactions involves instances in which metabolism by one or both Ps is influenced by the presence of the other enzyme. Interestingly, the degree of inhibition was shown to be dependent on the relative CPR ratio because at a 3: P ratio, no inhibition of 7-pentoxyresorufin dealkylation PROD was observed.
Although these results showed that one P could affect the function of a second P, the specific mechanism was not identified. This effect was much more pronounced at subsaturating CPR and approached additivity at saturating reductase. These results supported the concept that Ps could interact in a manner that modified their function, and raised the possibility that Ps exist in the membrane as subunits of complexes with unique functional characteristics.
In their study, CYP1A2 and CYP2B4 were labeled with different fluorescent labels that reacted with the sulfhydryl residues of cysteine, and the interaction between the enzymes was monitored by FRET in a low-concentration detergent solution. These results are consistent with the kinetic observations showing an activation of metabolism by CYP1A2 and an inhibition of that by CYP2B4 in dilauroylphosphatidylcholine membranes Backes et al.
Evidence for these functional interactions between CYP1A2 and CYP2B4 was not limited to reconstituted systems, but was also demonstrated using microsomal preparations from rabbit liver Cawley et al. These results show that the functional interactions not only are found in reconstituted systems, but also in the native milieu of the microsomal membrane.
The study presumed that the PP complexes would be disrupted by an alteration in the ionic strength of the solvent if electrostatic forces directed the interaction of the proteins. However, this synergistic stimulation was eliminated when the ionic strength of the medium was increased. In more detailed studies in which activities were measured as a function of CPR concentration, synergistic stimulation of EROD was shown to be observed only at subsaturating CPR, and the activity approached additivity at high CPR levels.
Interestingly, despite elevated electron transfer to cytochrome c, Pdependent monooxygenase activities measured at high ionic strength were not similarly elevated, demonstrating that the stimulation at subsaturating reductase was due to the P components.
The additive response observed at subsaturating reductase was unaffected by alterations in the ionic strength of the solvent. It may be tempting to conclude that these proteins do not form a heteromeric complex; however, these data only show that the function of the proteins is not affected by the combination.
It is still possible that the proteins form a complex that does not significantly alter their catalytic behavior for the substrates selected. Interestingly, these experiments were done with saturating CPR. CYP2C9 ratio of 3: Interestingly, where an equimolar CYP2C Again, the data is consistent with a Model 3 type of interaction.
Supporting the assumption that this change was caused by an interaction between the Ps, the effects were dependent on the concentrations of both CYP3A4 and CPR, and inhibition was detected even at saturating CPR.
These results rule out the possibility that these proteins are simply competing with limiting concentrations of CPR. Taken together, these studies provide ample evidence that different Ps can form complexes that actually change the catalytic rate reflected by an alteration in the rate constant for substrate turnover. This is likely due to a conformational change in one or both of the interacting Ps that allow either an enhancement in the rate of electron transfer or to some other catalytic step.
Demonstration that different P enzymes form heteromeric complexes leads to the possibility that Ps also have the potential to form homomeric complexes. Instead, the data were best fit to a model where homo- and hetero-oligomers of both CPR and CYP2E1 were present, in addition to the simple binary complexes. When the CYP2B4 solution was treated with 0. Thus, the data were consistent with an aggregate of CYP2B4 in which some enzyme units were protected from pressure-induced conformation changes in the absence of detergent.
Thus, the enzyme still demonstrated the properties of an aggregate in a lipid milieu. However, from the larger proportion of inactivated enzyme, it was suggested that the CYP2B4 existed as a tetramer in the proteoliposomes with three of the four units being sensitive to pressure changes. When oligomers were favored, CYP3A4 reduction was multiphasic; however, conditions favoring monomerization simplified the reaction, leading to monomeric reduction kinetics Davydov et al.
Thus, unlike the findings using the chemical reductant, dithionite, which showed complete reduction, only partial reduction of the P was observed with BMR. This could best be explained by the aggregation or complex formation between CYP3A4 molecules in a manner that prevented some of the Ps from interacting with BMR.
In fact, under conditions that were most similar to the microsomal lipid: First, there remains considerable uncertainty, over a range as large as mV, as to what the reduction potentials of heme-thiolate compound I intermediates really are But here it is still early days and there have been only a handful of measurements and estimates. There is, however, a basic difference between the productive cleavage of the substrate C-H bond in S-H by I and a non-productive, long-range electron transfer from an amino acid side chain.
In the productive pathway, a substrate proton arrives at the ferryl oxygen during the reaction. By contrast, the long-range electron transfer process would require a proton from some other source. Perhaps that proton is readily available through the water aqueduct leading to the P active site, but perhaps not.
Perhaps, also, an acidic proton from the water channel can activate the ferryl oxygen for substrate hydrogen abstraction 49 , Another major point of spirited debate has to do with the extent to which energy barriers for C-H bond scission, and thus the rates of these reactions, are affected by the exact electron configurations in oxidants such as I 51 , Although there has been progress recently in the preparation of synthetic ferryl species in both high-spin and intermediate-spin electronic configurations 53 — 57 , what matters is the arrangement of spin density at the transition state [SHO-Fe].
CYP research continues to be a rich, vibrant, and important field. Determining and understanding the reaction mechanisms of CYP substrate oxidations over the past several decades have greatly advanced a variety of fields.
Numerous spectroscopic techniques and diagnostic reaction probes have been applied to dissecting the mechanism. With this knowledge in hand, drug metabolism pathways can often be anticipated, weeding out poorly performing candidates early in the drug development pipeline. Reaction processes are being developed by using immobilized P and APO enzymes.
Also, new heme-thiolate proteins are being discovered. The author declares that he has no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The author thanks group members, collaborators, and colleagues in the field for discussion, inspiration, and many insights. No competing interests were disclosed. Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality.
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Close Copy Citation Details. Cytochrome P CYP enzymes are the primary proteins of drug metabolism and steroid biosynthesis. These crucial proteins have long been known to harbor a cysteine thiolate bound to the heme iron. Recent advances in the field have illuminated the nature of reactive intermediates in the reaction cycle. Similar intermediates have been observed and characterized in novel heme-thiolate proteins of fungal origin.
Insights from these discoveries have begun to solve the riddle of how enzyme biocatalyst design can afford a protein that can transform substrates that are more difficult to oxidize than the surrounding protein architecture.
The author s declared that no grants were involved in supporting this work. Pmediated biotransformations of the prodrug clopidogrel Plavix.
Analysis and discussion of the cytochrome P reaction cycle For all of these reasons, P enzymes have received sustained attention for decades. Cytochrome P hydroxylation mechanism. Typical active site of cytochrome P and aromatic peroxygenase heme-thiolate proteins. Concluding remarks Is the story over? Competing interests The author declares that he has no competing interests.
Acknowledgments The author thanks group members, collaborators, and colleagues in the field for discussion, inspiration, and many insights. F recommended References 1. Models and Mechanisms of Cytochrome P Action. Structure, Mechanism, and Biochemistry. Publisher Full Text 2. Ortiz de Montellano PR: Hydrocarbon hydroxylation by cytochrome P enzymes.
Alkane-oxidizing metalloenzymes in the carbon cycle. Mechanism of the Pyrocatechase Reaction. J Am Chem Soc. Publisher Full Text 5. Mechanisms of oxygen metabolism. Adv Enzymol Relat Subj Biochem. Imai Y, Sato R: Substrate interaction with hydroxylase system in liver microsomes. Biochem Biophys Res Commun. Discovery of the function of the heme protein P A systematic approach to scientific research. Publisher Full Text 8. A passion for Ps rememberances of the early history of research on cytochrome P Omura T, Sato R: A new cytochrome in liver microsomes.
Preliminary crystallographic data on cytochrome PCAM. High-valent iron in chemical and biological oxidations. Heme enzyme structure and function. Unusual cytochrome p enzymes and reactions. Spectroscopic features of cytochrome P reaction intermediates. Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step.
Hofrichter M, Ullrich R: Oxidations catalyzed by fungal peroxygenases. Curr Opin Chem Biol. Structure and chemistry of cytochrome P Oxoiron IV in chloroperoxidase compound II is basic: Rittle J, Green MT: Cytochrome P compound I: Thermochemistry of proton-coupled electron transfer reagents and its implications.
Formation of P450•P450 Complexes and Their Effect on P450 Function
Cytochromes P (CYPs) are the principal enzymes responsible for the oxidation of the wide and complex array of drugs and foreign chemicals to which the. Cytochrome P enzymes are essential for the metabolism of many dose, has recently been difficult to anticoagulate to a therapeutic level. One recent example of an unusual rearrangement in a P complex involves the P enzyme CYP (from Mycobacterium tuberculosis.