Heme alkylation

Ortiz de Montellano PR, Komives EA. Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450. J Biol Chem 1985 260(6) 3330-3336. 

Figure 9.17 Measurements of CO spectra before and after incubation with MBI. (a) CO measurement indicating partial loss of CO binding despite being an apoprotein adduct (b) positive heme adduct, ABT, showing heme alkylation and subsequent disruption of CO binding and (c) MBI, midazolam thought to result from apoprotein adduct also showing marginal loss of CO binding [198].

Further support for electron abstraction from nitrogen is derived from experiments on 1,4 dihydropyridine 138 with hepatic microsomes [96]. In the course of the incubation significant deactivation of the P450 enzyme was observed suggesting heme alkylation. Subsequent isolation and characterization of N-ethylprotoporphyrin IX indicated an ethyl radical transfer from the... 

In contrast, trans olefins are often poor substrates for CPO. When the double bond is far from the chain terminus (i.e., from r/.v-3-alkenes upward), allylic hydro-xylation accompanies the epoxidation. In addition, with terminal monosubstituted olefins, heme alkylation occurs, thus producing inactivation of CPO. 1-Alkenes can be profitably oxidized to epoxides by CPO only when they are not monosubstituted. A detailed description of the yields and e.e. for CPO-catalyzed epoxidation of olefins has been reported by Adams and coworkers [23]. 

P450s are generally less stable than peroxidases. Recently a naturally thermostable P450 (stable up to 85 °C) was identified and characterized [25, 100 -103], Both P450s and peroxidases are inactivated during catalysis, via heme alkylation by terminal olefins (see Section 10.8.3) and oxidative damage by peroxides. Eukaryotic P450s are associated with cell membranes and are therefore insoluble and difficult to use outside the cell. We believe that many of these limitations can be addressed by directed evolution. 

Ortiz de Montellano, E R., Mangold, B. L., Wheeler, C Kunze, K. L and Reich, N. O. (1983). Stereochemistry of Cytochrome P450 Catalyzed Epoxidation and Prosthetic Heme Alkylation, /. Biol. Chem., 258 4208—4216,... 

P-450 has been shown to catalyze epoxidation with retention of the olefin configuration (114). Ortiz de Montellano and co-woiicers have shown that heme N-alkylation accompanies epoxidation when terminal olefins are oxidized by P-450 (775). Further, the oxidation of 1,1,2-trichloroethylene is known to give trichloroacetaldehyde along with epoxide (776, 777). A mechanism that explains simultaneous epoxidation, heme alkylation, and halogen migration is depicted in Scheme XVI (777). In this process, initial electron transfer affords a transient rr-radical cation that can collapse with C-0 bond formation to give either radical or cation intermediates. 

Addition of the ferryl oxygen to the internal carbon of a terminal acetylene results, as already discussed, in prosthetic heme alkylation. Addition of the oxygen to the terminal carbon, however, is associated with intramolecular 1,2-shift of the terminal hydrogen to give a ketene (Ortiz de Montellano, 1986). The... 

Figure 6.17. Explanation of the dual pathways of olefin oxidation resulting in epoxide formation and heme alkylation in terms of the two-state hypothesis of P450 catalysis extensively described in Chapter 2.

Ortiz de Montellano, P.R., B.L.K. Mangold, C. Wheeler, K.L. Kunze, and N.O. Reich (1983). Stereochemistry of cytochrome P-450-catalyzed epoxidation and prosthetic heme alkylation. J. Biol. Chem. 258, 4208-4213. 

Raner, G.M., J.A. Hatchell, M.U. Dixon, T.L. Joy, A.E. Fladdy, and E.R. Johnston (2002). Regio-selective peroxo-dependent heme alkylation in P450BM3-F87G by aromatic aldehydes Effects of alkylation on catalysis. Biochemistry 41, 9601-9610. 

P450 is often irreversibly inactivated via covalent attachment of the catalytically activated inhibitor, or a fragment of it, to the heme group. A heme alkylation mechanism has been unambiguously demonstrated, in many instances, by evidence of equivalent activity and heme loss and the isolation and structural characterization of the modified hemes. It must be noted that in the absence of explicit evidence for heme adduct formation, an equimolar loss of enzyme content and heme does not unambiguously establish that heme alkylation is responsible for enzyme inactivation because alternative mechanisms exist for the catalysis-dependent destruction of the heme (see Section 3.4). It is also possible for a heme adduct... 

The only structural requirement for prosthetic heme alkylation by olefins is a monosubstituted double bond. Accordingly, ethylene, but not ethane, is able to destroy the P450 heme while... 

Ethylene, propene, and octene, all linear olefins, only detectably alkylate pyrrole ring D of the prosthetic heme of the phenobarbital-inducible rat liver P450 enzymes (Figure 7.15), but heme alkylation by the more globular olefins 2-isopropyl-4-pentenamide and 2,2-diethyl-4-pentenamide is less regiospecific ... 

The regiochemistry and stereochemistry of heme alkylation by olefin stereochemistry is preserved... 

Several mechanisms can be envisaged for heme alkylation that are consistent with the experimental data, none of which involves a concerted transfer of the oxygen to the rr-bond. Subsequent to possible formation of a charge transfer complex between the ferryl species and the olefin -ir-bond, addition of the oxygen to the Tr-bond could give a... 

P450-catalyzed oxidation of terminal acetylenes to substituted acetic acids (Chapter 6) is more prone to result in heme alkylation than the oxidation of terminal olefins. The structure-activity relationships for the acetylene reaction are similar to those for terminal olefins, except that there are fewer instances in which the reaction does not result in errzyme inactivation. For example, P450 is inactivated by phenylacetylene but not delectably by styrene ", and P450 is inactivated by internal acetylenes, albeit without heme adduct formation, but not by internal olefins 22 , Catalytic oxidation of the acetylenic function is required for enzyme inactivation and terminal acetylenes give heme adducts analogous to those obtained with terminal olefins - 259 jhe salient difference in the adducts obtained with acetylenes and olefins... 

Figure 7.16. The P450-catalyzed oxidation of a terminal acetylene partitions between formation of the ketene and heme alkylation. Which of these events occur is determined by the carbon to which the activated oxygen is added addition to the internal carbon (a) results in heme alkylation, and to the terminal carbon (b) yields the ketene. In the absence of the iron, the enol adduct tautomerizes to the ketone.

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