The degradation of haem by carbon tetrachloride: Metabolic activation requires a free axial coordination site on the haem iron and electron donation

Abstract

1. The ability of haem to catalyse the reductive activation of carbon tetrachloride (CCl₄) in vitro has been investigated under anaerobic conditions, using methaemalbumin (MHA) and either sodium dithionite or NADPH together with NADPH-cytochrome P-450 reductase (EC 1.6.2.4) as the reducing agents.

2. In the non-enzymic system protohaem and other non-physiological haem analogues underwent rapid and extensive CCl₄-depedent degradation, due to irreversible modification of their porphyrin tetrapyrrolic structure.

3. This mechanism of non-enzymic activation of CCl₄ by protohaem mimics that catalyzed by cytochrome P-450 in that it requires a free, reduced haem iron and electron donation and it is largely prevented by carbon monoxide.

4. H.p.l.c. analysis of ¹⁴C-haem after anaerobic incubation with CCl₄ and sodium dithionite gave radioactive products which eluted before and after haem, and exhibited significantly lower absorbance at 400 nm compared with authentic haem. When the products of CCl₄-dependent haem degradation were methylated and applied to silica for t.l.c., two non-fluorescent pigments were isolated, purified and partially characterized.

5. On incubation of haem with ¹⁴CCl₄ and sodium dithionite a 1:1 stoichiometry could be calculated for haem loss and ¹⁴CCl₄-derived adduct formation, indicating that, as with microsomes, the loss of haem may be the result of a typical ‘suicidal’ inactivation reaction where the same haem moiety is both the site of CCl₄ activation and the target of CCl₄ reactive metabolites.