Coupled oxidation vs heme oxygenation: Insights from axial ligand mutants of mitochondrial cytochrome b5

Document Type


Publication Date



Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b5 (OM cyt b5), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O2. The reaction proceeds via the formation of an oxyferrous complex (FeII-O2) that is reduced by hydrazine to a ferric hydroperoxide (FeIII-OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (FeIII-OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b5, to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of FeIII-H63V with H2O2 leads to the nonspecific degradation of heme, the incubation of FeIII-H63V with H2O2 results in the formation of meso-hydroxyheme, which upon exposure to O2 is rapidly converted to verdoheme. These findings revealed that although mesohydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O2 is reduced to noncoordinated H2O2, which reacts with FeII-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O2 molecule (FeII-O2) is reduced to a coordinated peroxide molecule (FeIII-OOH), which oxidizes heme to meso-hydroxyheme.

Publication Source (Journal or Book title)

Journal of the American Chemical Society

First Page


Last Page


This document is currently not available here.