Hemin is kinetically trapped in cytochrome b5 from rat outer mitochondrial membrane

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Cytochrome b5 from the outer mitochondrial membrane of rat liver (OM cyt b5) is substantially more stable to thermal and chemical denaturation than cytochrome b5 from the endoplasmic reticulum of bovine liver (microsomal, or Mc cyt b5). In contrast, the corresponding apoproteins have similar stability, suggesting stronger interactions between hemin and the polypeptide in OM cyt b5. Whereas complete transfer of hemin from bovine Mc cyt b5 to apomyoglobin at pH 5.2 takes less than 1 h, hemin transfer from OM cyt b5 is unmeasurably slow. Coupled with the previously reported 1:1 ratio of hemin orientational isomers in OM cyt b5, this finding suggests that the cofactor is kinetically trapped under physiologically relevant conditions. This conclusion is confirmed by 1H NMR studies which show that the hemin isomeric ratio changes when the protein is incubated for several hours at 68°C. Interestingly, the orientational isomer favored in OM cyt b5 is the form less favored in all other known cytochromes b5. (C) 2000 Academic Press.

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Biochemical and Biophysical Research Communications

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