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Both the books detail the structure and specific function of the cytochrome P-450. CYP-450 helps in the oxidation of organic substrates and in the detoxification process. The paper reviews its mode of function and how the spectral properties are affected as the electrons from Fe(III) or porphyrin ring move from lower to higher energy levels. While Gunter & Turner (1991) illustrate the use of metalloporphyrins as model for CYP-450, Williams et al. (2003) describes the crystal structure of CYP-450 in humans, in a state where it is bound with the drug warfarin.
P450 cytochromes are membrane bound mono-oxygenase enzymes and catalyze the reaction that involves transfer of oxygen atom to non-polar substrates. CYP helps in the oxidation of organic substances. A split is produced in 420nm Soret band if the enzyme binds with carbon monoxide. Bands are formed at 364nm and 450nm. P450 enzymes absorb light at wavelengths near 450 nm, identifiable as a characteristic Soret peak. It has been established that these haemoproteins are not cytochromes as they do not take part in the electron transport chain but rather are involved in oxygen transport. Binding with O2 produces spectral changes similar to binding with CO.
With NADPH as the electron source, the catalyst reduces oxygen to water while alkanes and arenes are hydroxylated and alkenes form epoxides. C-C bonds are cleaved in certain steroids by this enzyme. They exist in mammalian cells, insects, plants and bacteria. Liver in mammals contain the highest concentration of P-450 cytochromes. Steroids, fatty acids, leukotrienes, prostaglandins, solvents, pesticides, etc may act as substrates. However, it is believed to also aid in cancerous growth brought on by the free radicals generated during catalysis.
The hemoprotein contains a prosthetic iron-haeme cofactor bonded to two ligands axially and a proto-porphyrin molecule, in which the reactivity of iron depends on its variable oxidation states. Iron may exhibit two oxidation states by losing electrons from the two 4s electrons and six 3d electrons. Ferrous ion or Fe2+ result from losing two electrons from the 4s shell while losing three electrons, including one from the 3d orbital results in a ferric ion or Fe3+. Fe3+is much more stable that Fe2+ as it has a stable half-filled 3d orbital configuration. This property allows cytochrome P450 two readily undergo reduction as it accepts an electron from the haeme group.
In a mono-oxygenation cycle, the substrate gets attached to the ferric enzyme which exhibits low spin and as a result displaces the water molecule originally placed as an axially located ligand to the haeme core. The enzyme now attains a high spin state. This allows for easier reduction as a greater reducing potential is achieved. An O2 molecule, then binds to the haeme center to form an oxy-haeme complex. After a series of protonations and heterolysis of the O-O bonds, the catalyst is retrieved as the substrate undergoes oxidation during the cycle.
Isoenzymes of P-450 have been discovered with slightly reactivity and selectivity to substrates. Substrates can initiate the production of specific isoenzymes and this is evident from the fact that alcoholics who suffer from liver cancer have high levels of cytochrome P-450j the production of which is induced by ethanol. Polymorphism is a known characteristic of this enzyme and this causes variations in effects of the increase in cytochrome levels like in lung cancer risk in chain smokers.
The primary function of CYP 450 is apparent when one considers that the group of enzymes located on the endoplasmic reticulum, are important for the metabolism and transformation in drugs. The gene coding for cytochrome is known to have existed for over three and a half billion years. Therefore, the fact that they help in drug metabolism is a possible new role that has been undertaken by CYP 450. P-450 aids in detoxification of endogenous compounds that have been consumed as food. This explains the higher concentrations of this enzyme in liver and small intestine.
These haemoproteins are not like regular cytochromes and they do not play any major role in electron transport. Present on cell membranes, they catalyze hydroxylation of C-H bonds by what is known as the mono-oxygenase reaction:
RH + O2 + 2 H+ + 2 e- = ROH + H2O.
The insertion of OH group in the hydrocarbon prevents damage by drugs, steroids and pesticides.
The target compounds become more water soluble with the addition of the hydroxyl group and get eliminated more easily.
Structure of P-450 cytochrome includes a hemin group i.e. Fe (III) center that is coordinated to thiolate in cysteine residue. A stable octahedron is formed with the addition of a water molecule.
The substrate, RH, binds to the enzyme in a hydrophobic pocket on the enzyme that is close to the active site, near the haeme group. The active site undergoes conformation in its bound state by displacing water molecule. Heme iron may change state from low spin to high spin. The spectral properties are affected and can bring about two types of difference spectrum. H2O molecule is lost as Fe (III) moves from a lower state to higher state of spin.
The change in the electronic state allows reductase like cytochrome P-450 reductase to transfer electron from NAD(P)H. Electron transfer from redox protein reduces Fe (III) to Fe (II) which then binds to O2. The oxy form has lower state Fe (III). O2 binds with a covalent bond to haeme at the distal axial coordination position.
A second electron is transferred with the uptake of two H+ ions to form a ferryl complex between Fe (IV) and oxygen bound with double bonds (FeIV=O), and H2O. A related one-electron oxidation step of porphyrin ring takes place to form a ring radical anion which will later form the ferryl complex. The anion at this point attacks the substrate and oxidizes it. Oxygen is inserted, ROH is lost and H2O molecule is taken up. This completes the cycle. The neutral O-unit is then free to leave the iron porphyrin to reduce Fe (IV) to Fe (III).
Rabbit liver has been used to study phenobarbital induced P-450LM2. P-450cam or bacterial camphor-hydroxylating enzyme from Pseudomonas putida has a structure resembling a triangular prism and about 30 Å thick. It has 12 helical segments arranged in three layers with a proto-porphyrin IX sandwiched between the layers. The binding site for camphor is surrounded with hydrophobic pockets and the substrate is placed about 4 Å from the porphyrin plane beside the O2 binding site. The presence of a reversible cap allows the substrates to bind and protects them from the external surroundings.
The porphyrin is attached to the enzyme by a single amino acid cysteine residue. The sulfur atom is in touch with oxygen and nitrogen atoms that form the backbone of the helical peptide sequence of four proteins. A network of five water molecules, held together by hydrogen bonds, gives a hydroxide-like character to the substrate-free enzyme.
Using recombinant DNA technology, homologous regions on the P-450 proteins have been discovered. All P-450 cytochromes, in eukaryotes and prokaryotes, have the same active site and reaction cycle. Recombinant DNA technology has been employed in the synthesis of protein in active form and enzymes can be modified through mutagenesis specific to particular sites. This could be used to manipulate the regiospecificity of hydroxylation for several substrates. Under suitable conditions, P-450 cytochromes can be made to function as peroxidase, catalase, chloroperoxidase, etc because of the protoporphyrin IX prosthetic group similar to globins.
Electron transfer occurs on the enzyme but for compounds to effectively hydroxylate and epoxidize substrates, porphyrin rings have to be substituted with aryl meso residues to protect from other reactions.
Cytochrome P450 in mammals recognize and break down xenobiotics (Williams et al, 2003) like drugs and pollutants. Known isoforms of p-450 in humans are CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. These help in the metabolism of 90% of drugs used currently (Williams et al, 2003). While studying the structure of human P-450, CYP2C9 has been found to interact with warfarin in a newly discovered binding pocket. Warfarin is an anti-coagulant. This also suggests that CYP2C9 accomodates multiple ligands at the same time. The B-C loop present in CYP2C9 aids in specificity to particular substrates.
A water molecule is located 7Å above the haem iron and bonded with hydrogen bond to threonin, Thr 301, that takes part in proton transfer path. CYP2C9 prefers small acidic lipophilic compounds that point towards the presence of basic residues in protein active site. This is the hypothesis of anionic-binding site. However, two acidic residues have been found in the active site of CYP2C9. In humans, isoforms CYP2C9 and CYP2C19 differ by 43 residues out of a total of 490 but only one at which non-conservative substitution occurs. Residue 99 is isoleucine in CYP2C9 and histidine in case of CYP2C19.
Structure of CYP450- CYP2C Surface of the active site of CYP2C9
The active site as is seen in the image is large, approxiamtely 40 Å3 and can accomodate compounds larger than S-warfarin or multiple ligands if their sizes are comparable to S-warfarin without substantial conformational movement. With S-warfarin bound in this location , haem group is still available to metabolize other substrates. This is so because S-warfarin is placed quite distant for hydroxylation to occur. The compound has to move towards the primary recognition site.
More work on P450 was done by Sawyer and co-workers in 1988 in isolating a green compound (formed by the reaction of [tetrakis(2,6-dichlorophenyl)porphinato]-iron(III)perchlorate,FeTP 2,6-Cl PClO4, m-chloroperbenzoic acid and ozone in acetonitrile ar -35C) which acts as an competent catalyst for stereospecific epoxidation of alkenes.
Traylor et al pointed at the correlation between oxidation potential and catalyst activity. The model catalyst was found to oxidise substrates with a potential of less than 1V, which also is the same for Horse-radish Peroxidase. These function as effective monooxygenase models because of the presence of meso positions on the porphyrin ligand. That is a principle point for spin accumulation when electron is lost from the porphyrin (Gunter & Turner, 1991)
Manganese porphyrins activate hydrocarbons with biomimetic oxidation with the generation of high valent manganese oxo complexes. Hill and Hollander in 1982 were able to spectroscpically study the structure of Mn(V) nitrido porphyrin complex and revealed the true oxidation state to be lower than the normal state due to electron delocalization of the N3- and O2- ligands.
The results of the P-450 model studies indicate multiple possible pathways for catalytic reactions. These pathways are pre-determined by the topology and geometry of the catalyst and substrate that has to undergo oxidation. It is also evident that single-electron alkene oxidation to form epoxide is process independent.
P-450 is used to study the effects of drug metabolism in humans. Drugs undergo biotransformation after which they are eliminated from the system. The effects of drugs are observed because some drugs increase or decrease the activity of CYP by inducing or inhibiting the enzyme. This helps in adjusting drug dosages by studying P-450 and helps prevent toxicity in the body as accumulation levels of drugs are easily researched. Therefore, the scope and use presented by CYP450 is tremendous and can definitely help humans in therapeutic and pharmaceutical fields.
Gunter and Turner, in their article explain the structure and function of CYP-450 with the use of metalloporphyrins like manganese porphyrin. In my opinion, the authors have been able to support and confirm the process of abstracting hydrogen and the following recombination mechanism in the Fe and Mn model catalysts. In the article with human cytochrome bound with warfarin, the researchers report the presence of a new binding pocket on CYP2C9 to accomodate multiple ligands. This is a possible mechanism to explain complex drug-drug interactions.