Abstract
1. The molecular mechanism and the reactive species involved in the inactivation of microsomal cytochrome P-450 during the reductive metabolism of carbon tetrachloride (CCl[4]) were studied. Results obtained with spectrophotometric, fluorimetric and chromatographic methods and using strictly anaerobic conditions indicate that the prosthetic group of the cytochrome, haem, is both the site and the target of CCl4 activation. 2. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when CCl[4] was incubated anaerobically with NADPH- or sodium dithionite-reduced rat liver microsomes. A rapid loss of haem was also observed during reductive incubation of CCl[4] with soluble haem preparations (methaemalbumin) in the absence of the apoprotein. In both cases the loss of haem was accompanied by an equimolar loss of its tetrapyrrolic structure and was prevented by carbon monoxide. Similar results were obtained using halothane as a suicidal substrate. 3. A dichlorocarbene-cytochrome P-450 ligand complex was found to be responsible for the difference spectrum obtained on addition of CCl[4] to anaerobically reduced rat liver microsomes. A molar extinction coefficient of 56.2 for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene did not prevent, however, the CCl[4]-dependent loss of microsomal haem indicating that the carene is not involved. 4. Inactivation of haem by CCl[4], both in microsomes and the non-enzymic system is a typical suicide reaction which follows saturable, pseudo first-order kinetics. A partition ratio of 26 was calculated between molecules of CCl[4] metabolised and molecules of cytochrome P-450 haem lost. The loss of haem in the chemical system is due to covalent binding of CCl[4] reactive metabolites in a 1:1 stoichiometry.