http://www.unc.edu/courses/2009spring/envr/740/001 slide 22Begin 03/19/09The superoxide anion can dissociate, which leaves the enzyme in the resting state, but has released a progenitor of ROS. This process is referred to as futile cycling of P450, because it involves the consumption of O2 and reductant without generating any product from the substrate.If the cycle continues, reduction of the heme·O2·substrate complex by a second electron leads to a peroxoiron complex, formally O22- or looked at another way, the dianion of hydrogen peroxide. Recent work supports protonation of the peroxoiron complex to generate a hydroperoxo complex, formally a complex of the mono anion of hydrogen peroxide, called cpd 0. Studies in model systems suggest this complex could oxidize unsaturated substrates predominantly by epoxidation. However, interest in cpd 0 has waned, and an alternative pathway, which is the one initially proposed and which has always represented the major reaction pathway, is addition of a second proton which results in the release of water with the transient formation of an extremely reactive monooxygen complex called compound I, that is oxidized by two electrons above the ferric resting state of the enzyme. The oxygen atom is rapidly transferred to the substrate, followed by release of the oxidized substrate and return of the cytochrome P450 to the resting state. Compound I can hydroxylation carbon and epoxidize double bonds. Investigators have used site-directed mutagenesis (mutating specific amino acid residues) to perturb the hydrogen-bonding network and proton sources at the active site of cytochrome P450 to alter the balance of the two pathways for oxidation of specific probes having both a double bond and an activated C–H bond. These experiments are interesting in terms of their implications for the effects of single nucleotide polymorphisms, and we will discuss them again at in that context.In support of the cpd I pathway is the fact that the enzyme will perform oxygen transfer in the absence of the normal cofactors (O2 and an electron donor) when a monooxygen donor such as a peroxy acid or hydroperoxide is present as a monooxygen source:Repeat [cytochrome P450 cycle]That is indicated by the “shortcut” in the cycle, which is called the peroxide shunt. A bacterial 1cytochrome P450, P450cam, has been crystallized with the camphor substrate bound at the active site. By a very clever technique, a crystal structure of a monooxygen transient was obtained:[ENVR 740 Part 4, crystal structure of P450cam I, distal view]The view on the overhead looks down on the distal face of the porphyrin (distal is the face opposite the Cys ligand where the substrate is bound and proximal is the face to which Cys is coordinated). Note that the pathway into the distal pocket at the reactive site is rather restricted. This is a characteristic of bacterial cytochromes P450, which are extremely selective towards substrates. The next overhead[OH 20; ENVR 740 Part 4, crystal structure of P450cam I, edge view]shows a view more from the edge of the heme. Here, it is easy to see the coordinated cysteine, the oxo unit and the steric relationship between the oxoferryl unit and the camphor substrate. It isclear from the coordination geometry, that transfer of the oxygen atom will be both highly regioselective (a specific site on the substrate is oxidized) and highly stereoselective if the product is chiral.To obtain this structure, a one-electron reducing agent was diffused at low temperature into crystals of resting P450cam which had been crystallized with the camphor substrate to give the Fe2+ form of the complex, followed by diffusion of dioxygen at high pressure to generate the ferrous-dioxygen-camphor ternary complex. The ternary ferrous P450·O2·camphor complex was then reduced an additional time with hydrated electrons generated by x-irradiation of the frozen aqueous sample to give the iron oxo transient shown in the overheads. There is ambiguity as to whether the iron-oxo complex generated under these conditions is at a level of 1 or 2 electrons above the resting state. The next overhead explains the manner in which the ambiguity about oxidation state of P450 arises.Slide 21[OH 21, ENVR 740 Part 4, homolysis/heterolysis of hydroperoxo complex]2In the top pathway, the O-O bond of the hydroperoxy complex is cleaved homolytically (i.e., each oxygen departs with one unpaired electron), yielding a hydroxy radical and an iron(IV) porphyrin, which is oxidized by one electron above the resting ferric state of the enzyme. This species is called cpd II. In the bottom pathway, the O-O bond is cleaved heterolytically, releasingone of the oxygens (formally) as HO- along with cpd I, which is oxidized by 2 electrons above the resting state.Efforts to purify and characterize cytochrome P450 proteins from eukaryotic organisms have revealed that there are a very large number of isoforms of this enzyme, each having its own substrate and stereochemical selectivity. The next overhead illustrates members of the cytochrome P450 superfamily in the genome of the fruit fly.[OH5, ENVR 230 Part 5, Melanogaster P450 superfamily](Don’t even think of trying to sort this out. The overhead is meant to illustrate the huge size of the superfamily.) Until recently, there had been no structures of eukaryotic cytochromes P450, however, within the past few years, structures have been obtained from “engineered” recombinant gene products by truncating the proteins at membrane anchoring domain and crystallizing the remaining soluble truncated protein. An example of such an “engineered” protein structure is rabbit CYP2C5:[OH6; ENVR 740 Part 4, structure of CYP2C5 from rabbit]This view shows the distal pocket. It is immediately evident that the protoporphyrin is considerably more accessible than that of P450cam. This is in accord with the function of mammalian cytochromes P450 to accommodate a variety of substrates, including relatively large molecules. The active site of CYP2C5 can accommodate progesterone, benzo[a]pyrene and estradiol.[OH; ENVR 740 Part 4, structures of P450cam, CYP 2C5 and CYP3A4]3The next slide shows a comparison of P450cam, CYP2C5 and human CYP3A4 in similar orientations to illustrate relative accessibility of the distal face of the porphyrin. Critical amino acids in the proximal and distal pockets; e.g., Cys coordinating the iron in the proximal pocket and residues involved in the network