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The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation
Abstract:
Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant.
Insights
Cytochrome P-450 enzyme is destroyed by certain dihydropyridine substrates through a radical mechanism. This destruction involves the transfer of an alkyl group to the heme, indicating P-450 can act as a one-electron oxidant.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Organic chemistry
Background:
- Cytochrome P-450 enzymes are crucial in metabolizing xenobiotics.
- Certain substrates can inactivate Cytochrome P-450 during catalysis.
- The mechanism of this inactivation is not fully understood.
Purpose of the Study:
- To investigate the mechanism of Cytochrome P-450 destruction by 4-substituted 1,4-dihydropyridine substrates.
- To correlate enzyme inactivation with substrate structure and radical stability.
- To identify the reactive intermediates and adducts formed during the process.
Main Methods:
- Catalytic oxidation of dihydropyridine substrates using purified Cytochrome P-450.
- Analysis of reaction products, including heme adducts (N-alkylprotoporphyrin IX derivatives).
- Spin-trapping studies using alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone to detect radical intermediates.
- Investigating the role of hydrogen peroxide in the inactivation process.
Main Results:
- Cytochrome P-450 destruction correlates with the radical stability of the 4-substituent.
- N-alkylation of heme occurs with 4-ethyl, 4-propyl, and 4-isobutyl analogues, forming isolable adducts.
- Ethyl radical formation from DDEP (4-ethyl analogue) was confirmed by spin-trapping.
- Hydrogen peroxide contributes to ethyl radical formation and enzyme self-inactivation.
- The oxidation process proceeds via one-electron steps.
Conclusions:
- Cytochrome P-450 mediated inactivation by these dihydropyridines involves substrate radical formation.
- The enzyme acts as a one-electron oxidant in the N-alkylation of heme.
- The stability of the 4-substituent as a radical is a key factor in the rate and extent of enzyme destruction.