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Isolation of Enzyme
The isolation of Cytochrome P450 is often performed in order to study its enzymatic structure along with its catalytic involvement in oxidative reactions. It can be completed via a series of various column-assisted purifications at low temperatures (~4oC). To initiate the purification, the enzymes must be extracted from microsomes obtained from biological samples. Microsomes are obtained from CYP-containing species such as humans and Pseudomonas putida. CYP 2C19 can be found in the human liver, and the following information outlines how this specific enzyme can be isolated. The microsomes are solubilized in a desired buffer. An ideal amount of the CYP enzyme can be solubilized in sodium cholate, when the sodium cholate solvent is at 3 times the concentration of the protein sample. The solubilized solution is centrifuged, and the, the supernatant that contains the microsomes of interest. The separation from other biological particles can be done on a hydrophobic column, such as aminooctyl-sepharose. At equilibrium, CYP 2C19 can be eluted from an aminooctyl-sephradose column with an appropriate solvent mixture of anionic cholate solution and non-ionic surfactants. To determine the elution profile, immunoblot assays are done, using the wavelength of 417 nm to detect the enzyme in the column fractions. Further separation can be done on weak anionic exchange columns. This is often completed using DEAE-sepharose column series, where the enzyme can be eluted with a buffer containing a positively charged surface containing glycerol, EDTA, and a non-ionic surfactant. On a cation-exchange column, CYP purification can be achieved if the exchangers of the column exist as either carboxymethyl cellulose or DEAE-cellulose; the CYP enzyme would take a negative charge in this case. The elution of Human CYP can then be acheived with an increasing concentration of an ionic salt, such as NaCl, as the mobile phase. To produce optimal isolation, dialysis is done on each fraction (containing CYP) in between each elution. This ensures that small unwanted salt molecules are removed from the protein sample, otherwise the protein sample will run down in the following separation.

Chromatography over hydroxyapatite as a column has shown to be effective. Biochemists have shown to attain CYP elution with small concentrations of a potassium phosphate buffer. To remove trace CYP proteins from the eluted samples, immunoadsorption chromatography is usually applied, using a column equipped with an antibody that is specific for the undesired CYP. Ultrafiltration is used to further concentrate each sample of CYP that is acquired. In analyzing the CYP content of the samples, immunoblot analyses developed with polyclonal or monoclonal antibodies that are specific to the desired CYP is practical.

Technique
Time-resolved Crystallography can be used to determine the crystal structures of the intermediate enzyme substrate complexes involved in the Cytochrome P450 catalytic pathway. This application produces “time-lapse” images of crystalline structures, and is assisted by freeze-trapping the complexes. X-ray data sheets are generated for examination, which entails results pertaining to bond lengths and positions of the components within various ferric P450 complex crystals. Protein crystallography has been enhanced to allow for the analysis of moderately stable intermediate complexes at cryogenic temperatures. This technique is of common amongst many researchers as enzyme catalytic pathways have become of avid interest. Schlichting and his co-workers were able to apply this technique to the Cytochrome P450 enzyme found from the Pseudomonas putida species.

Heme Group
·        Thiolate of Cys357 is affixed to the iron of the heme group via covalent bonding.

·        Described to be ruffled

·        Five-coordinate iron atom extends out of the plane at which the porphyrin lies by 0.3 ± 0.2 Å

Camphor
·        Located in the distal heme pocket.

·        Affixed be a hydrogen bond between the oxygen of the camphor carbonyl, and the hydroxyl group of Tyr96 at a bond length of 2.9 ± 0.2 Å.

·        Shifted from the vacant sixth ligand position

Thr101
·        Involved in a Hydrogen bond with the carboxylate ion from the D pyrrole (2.7 ± 0.2 Å).

·        In comparison to Protein Data Bank (PDB) [code 2CPP], the side chain seems to be rotated.

Heme Group
·        Salt link amongst the 6-propionic group, Arg112, and His355 remains unchanged.

·        Iron and thiolate of Cys357 are still hydrogen-bonded (2.2 ± 0.2  Å).

·        Sixth ligand position is still vacant.

Bonded Molecular Oxygen
·        The oxygen molecule is bound to the iron of the heme group at a hapto number of 1 (1.8  ± 0.2  Å).

·        The distant oxygen is situated towards Thr252 of the complex

·        Molecular oxygen is bent in structure

Camphor
·        Undergoes displacement during ligand addition of molecular oxygen, away from heme group.

·        In close proximity of the diatomic sixth iron ligand.

Heme Group
·        Significantly flatter.

·        Iron and axial thiolate of Cys357 bond at (2.3 ± 0.2  Å).

·        Iron atom above porphyrin plane.

New Ordered Water Structures: WAT901, WAT902
·        Become present simultaneously as molecular oxygen bonds to complex.

·        WAT901 is situated in the “groove” of the distal I helix.

·        WAT901 is n proximity of the O2 ligand.

·        WAT901 hydrogen bonds with the nitrogen of the amide group in Thr252, which is rotated towards the heme pocket.

·        Interactions with Val247, and Gly248, seems to stabilize the WAT901 molecule.

·        WAT902 near to the hydroxyl group of Thr252.

·        WAT902Located 3.4 ± 0.2  Å away from WAT901, 2.0 ± 0.2  Å from WAT687, 2.5 ± 0.2  Å from the carbonyl oxygen of Gly248, 2.7 ± 0.2  Å from the amide nitrogen of Val253,   and 3.8 ± 0.2  Å from WAT556.

Water Chain
·        WAT687, WAT902, WAT566, and WAT523 accumulate to form a closed water chain that stretches from Thr252 towards Glu366.

Carbonyl Oxygen of Asp251
·        Underwent a 90o flip towards Asn255, and engages in a bond formation with it’s side chain amino group.

·        This causes the Asn255 to shift, and conformational change to the backbone atoms of Asn255 and Thr252.

Distal I Helix
·        Hydrogen bond formed with the hydroxyl group of Thr252.

·        Widened due the condition of the Hydrogen bond.

Other changes near Active Site
·        Conformation change in side chains of Thr181 and Leu244.

Heme Group
·        After the cleavage of the Molecular Oxygen bond, the Iron is left bonded to a singular oxygen atom.

·        Bond to thiolate remains nearly unchanged.

·        The oxyferryl oxygen is described to have an occupancy of 60%, and a temperature factor of 13 Å2.

Single Oxyferryl Oxygen
·        1.65  ± 0.2  Å bond distance with the iron of the heme group.

WAT903
·        Occupancy: 70%

·        Temperature Factor: 26 Å2

·        Thought to be the released water molecule upon cleavage of the molecular oxygen.

·        Situated near the oxyferryl oxygen, the hydroxyl group of Thr252, and the carbonyl of Gly248  bond distances from 2.5 ± 0.2 to 2.9 ± 0.2 Å.

WAT901 and WAT902
·        Absent or disordered

Camphor
·        As the distant oxygen is cleaved off, the camphor ligand shifts above the plane at which the porphyrin lies (± 0.2 ± 0.2  Å towards the heme iron), allowing relief from steric constraint.

Asp251 Carbonyl
·        Returned (flipped) back to its original arrangement as seen in the ferric P450 complex

Oxygen Activation
The cleavage of the molecular oxygen substrate is initiated by a proton transfer; this idea is supported by the crystal structures of P450 (obtained from Pseudomonas putida) found by Schlichting et al. Schlichting and co-workers found that the pathway for this transfer associated with a water chain anchored by Glu366 residue. The conformational changes in the protein backbones and side chains near the active site, essentially generates a proton shuttle that operates via water molecules. The purpose of Asp251 is to stabilize the water molecule, WAT901, in collaboration with Thr252, which rotates its amide nitrogen closer to the WAT901 molecule. The Thr252 also connects with the molecular oxygen substrate, in addition to WAT901 and WAT902. The rearrangement of the distal I helix, upon the bonding of molecular oxygen, accommodates the binding of WAT901 in it’s “groove”. WAT901 and WAT902 are suggested to be used as the proton donors in the proton transfer mechanism.