Abstract
The reaction of purified rabbit liver microsomal P-450 isozyme 2 with 4,4′-dithiobis(2-nitrobenzoate) (DTNB) exhibits first order kinetics and results in the modification of a single thiol, but causes no net loss of the native ferrous-carbonyl spectrum. Inclusion of both phospholipid and a tight-binding nitrogenous ligand, 1-benzylimidazole, in the reaction medium produces a burst-phase of DTNB modification, but the stoichiometry remains one thiol modified per polypeptide chain. The site of isozyme 2 rapidly labeled by DTNB and by monobromobimane, a fluorescent reagent for thiol groups, was shown to be Cys 152. Results obtained strongly suggest that Cys 152 does not provide the proximal thiolate ligand to the heme iron atom. Since Cys 152 represents one of the two highly conserved cysteine-containing regions in the P-450 cytochromes, it appears likely that the other region, containing Cys 436 in this rabbit cytochrome (corresponding to Cys 355 in bacterial P-450 cam, Cys 436 in rat P-450 b or e, Cys 461 in rat P-450 c, Cys 456 in rat P-450 d or mouse isozyme 3, and Cys 458 in mouse isozyme 1) is the source of the thiolate ligand to the heme.
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