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Cytochromes, as specified by the US National Library of Medicine, are heme proteins, whose characteristic model of action involves the transfer of reducing agents via the reversible change in oxidation state of the heme group. Within such enzymes, Fe(II) and Fe(III) are the principle oxidation states of the heme group. They are typically membrane bound, and the families of cytochromes are known by the wavelength range of their reduced alpha-absorption bands, as seen by UV/Visible spectrum spectroscopy.
Cytochromes P450 are a well-established superfamily of enzymes, essential to biology and the continuation of life as we know it. Their range of functions extends from the hormonal regulation of metabolism, through to reproduction and evolution. As stated by Lewis, (2005) this catalytic superfamily also have significant input into medicine, with impact being made in the fields of cancer, diabetes, hepatitis and surgical trauma.
Due to such critical involvement in life processes, many different types of cytochrome p450 are available for study, and as discussed by Stout,( 2004), many different conformations of the enzyme are available. It is understanding how these differing conformations, and their inherent effects upon biological reactions, which give us insight into the importance of cytochromes P450. The function and catalytic cycles of cytochrome P450, are dependant upon the structure of the enzymes, , as stated by Ortiz de Montellano, (2005).
The functions of the cytochrome P450 family are to catalyze the oxidation of many different hydrophobic substrates, through the changing oxidation state of the central Heme group. As shown by Hasemann, et al., (1995), the number of sequence identities between P450 enzymes, are generally low, meaning that the interaction between structure and function can be unclear. The most widely studied cytochrome P450, catalyzes the stereospecific reaction of the hydroxylation of monoterpene camphor, a bicyclic molecule with a general formula of C10H16O. This is in keeping with the way in which all cytochrome P450's catalyze reactions which cover a diverse range, and include among them the hydroxylation of aliphatic and aromatic compounds, epoxidations and dealkylations. This broad range of functions brings specific problems to defining the function of cytochromes P450's. As shown by Stout, (2004), a central problem to ththis definition of function for the cytochrome P450 family, is the very diverse range of reactions the enzymes catalyze, with their behaviour being akin to that of antibodies, where a singular class of proteins can ligate with a diverse range of ligands,while simultaneously maintaining their specificity.
Figure 1: The formation of steroid hormones and cholesterol within the human body, showing key substrates, products and essexntial members of the cytochrome P450 family. As can be seen, CYP51, CYP17 and CYP21 play crucial roles in the conversion of substrates to the formation of the final product.
Cytochrome P450's oversee the biosynthesis of cholesterol and steroid hormones within the body, as shown by fig. 1., while also being crucial to the interaction of drugs within the body, accounting for around 75% of drug metabolism. This is key, as many drugs either increase or decrease the activity of different cytochrome isozymes, depending on if the drug is activating the biosynthesis of an isozyme, or acting in a manner which directly inhibts the activity of the cytochrome P450 involved. This creates issues with the clearence of drugs, and can affect the metabolism of drugs, bringing about an adverse drug interaction.
The earliest P450 structure to be resolved was P450cam , which was solved in 1987. The heme, which is found in all cytochromes, exists within a hydrophobic environment, orientated nearly parallel to the surfaces of the L & I helices. Cytochrome P450s, have a helix rich right hand side, and a beta sheet rich left hand side. In total 14 Î±-helices and 5 mixed Î²-sheets are present. Across the superfamily, a consistency in structure is found. This consistency in tertiary structure, across the entirety of the P450 family, indicates that all portions of the molecule contribute to the coordination of the heme, substrate binding and the recognition, interaction or catalysis of other proteins or the membrane. An alteration within the primary or secondary structures of the protein enable the specificity of the cytochrome P450's to be altered. Stout, (2004), shows this by explainging how the change in the FG loop, within Cyp 119, which folds over the active site, differs depending on its interaction with differing inhibitors.
The heme group, which is at the centre of the protein is a 5-coordinate heme, as the cytochrome P450 catalyses the oxidation of a given substrate. There exists a widespread division, as stated by Smith, et al., (1994), of P450-containing monooxygenases systems into two main types, bacterial/mitochondrial (type I) and microsomal (type II). Mitochondrial P450 reactions contain three compounds: an FAD containing flavoprotein, either NADPH or NADH-dependent reductase, an iron-sulphur protein and cytochrome P450. The microsomal cytochrome P450 reaction contains two components; NADPH-cytochrome P450 reductase, a flavoprotein containing both FAD and FMN, and P450. Cytochrome P450 reductase appears to be a protein consisting of two domains which are homologous to ferredoxin; NADP+ reductases (FAD domain) and flavodoxin (FMN domain).
All the P450 reactions share common structural and functional domain architecture. By domain we mean polypeptide existing as an independently folding unit and possessing a certain function. Hence there are no fundamental differences between the protein domain and the individual protein component and all the P450 reactions can be considered as three-domain reactions; NADH- or NADPH-dependent FAD containing reductase (FAD domain),an iron-sulphur protein, found in a three component reaction, or FMN-binding domain and cytochrome P450.
An intermediate electron donor component might be presented by one of the functionally interchangeable proteins: lavodoxin-like, ferredoxin or cytochrome b5. It is indicated though, as shown by Hasemann, et al., (1995), that the structure of the cytochrome P450 has some bearing upon its funcionality and its substrate affinity.
Figure 2: Resolved structure of a cytochrome from the P450 family. The centre of the protein contains the heme group, with the adjacent Î±-helix and Î²-sheet showing at the top of the diagram.
2e- . 2H+For the majority of cytochrome P450 reactions, a general formula can be constructed, which is as follows:
P450RH + O2 ROH + H2O
Within this reaction RH represents the substrate which is being hydroxylated during the course of the reaction. The entire catalytic cycle can be broken down into five distinct stages, as shown by figure 3.
Figure 3: The catalytic cycle of cytochrome P450's, showing the distinct stages the reaction undergoes. The first (1) is the initial substrate binding, the second (2) is the initial reduction, the third (3) is the oxygen binding, the fourth stage (4) is the second reduction, while the fifth stage (5) is the product formation.
The first stage within the reactions of cytochromes P450, is the binding of a substrate. It is known that within the resting state of the enzyme, as stated by Lewis, (2005), it is mainly in a low-spin ferric form with water occupying the central, heme pocket. It is thought that there is one water molecule ligating the heme molecule, with ligation occurring at its distal face. The substrate binding is very rapidly occurring, with a high enzyme-substrate affinity.
The second stage involves the reduction of the heme group, from its Fe3+ oxidation state, to its Fe2+ oxidation state. In vivo cytochromes P450 are typically membrane bound, with the case of humans being bound to the endoplasmic reticulum, alongside NADPH Cytochrome P450 Reductase, which donates electrons to the reduction of the heme group.
The third stage is the acceptance and binding of O2. At this stage, active site has both the substrate and the molecular oxygen ligated to it. The heme group still remains within its Fe2+ state. An electronic rearrangement takes place, switching an electron onto the molecular oxygen. The iron then returns to its Fe3+ state. This creates an oxycytochrome P450 complex.
At stage 4 in the catalytic cycle, another electron is donated from the neighbouring NADPH cytochrome P450 reductase, creating with the bound oxygen a superoxide O22-. Cytochrome P450 then rapidly undergoes electronic rearrangement once again, cleaving the O-O bond, freeing O to react with the presence of 2 H+ to form water.
Stage 5, the product formation, is stated by Lewis, (2005), to be the least understood stage of the P450 catalytic cycle. While a number of models for the breakdown of the iron peroxy complex have been suggested, the common thought, as stated by Lewis, (2005) is the formation of an Fe(V)O intermediate. After the breakdown of this intermediate though, the reaction releases water and the oxygenated metabolite, and the cytochrome P450 is available to catalyse another reaction.
In conclusion the formation of the substrate bound oxycytochrome complex is dependent upon two factors. The first is the presence of substrate, which in line with the structure of the P450 involved, has specificity to the enzyme, and the second is the presence of an electron donating agent, to enable the reduction of the substrate to occur. The hydroxylation of the substrate allows for drugs to become safely water bound and excreted from the body, and the specificity of the P450 enables certain drugs to be targeted, akin to the action of the azole family of anti-fungals.
Once the final stage of the catalytic cycle has taken place, the readiness of cytochrome P450 to take part in further reactions can be of beneficial use to the speed at which a drug may take effect upon the body if targeted to the active site of a given cytochrome P450. Alongside this the protein is membrane bound, normally within the mitochondria or smooth endoplasmic reticulum. This location means that lipid soluble drugs, targeted at specific cytochromes can be delivered right to the heart of target cells.