Cytochrome b(5) from the outer mitochondrial membrane of rat liver (OM cyt b(5)) is substantially more stable to thermal and chemical denaturation than cytochrome b(5) from the endoplasmic reticulum of bovine liver (microsomal, or Mc cyt b(5)). In contrast, the corresponding apoproteins have similar stability, suggesting stronger interactions between hemin and the polypeptide in OM cyt b(5). Whereas complete transfer of hemin from bovine Mc cyt b(5) to apomyoglobin at pH 5.2 takes less than 1 h, hemin transfer from OM cyt b(5) is unmeasurably slow. Coupled with the previously reported 1:1 ratio of hemin orientational isomers in OM cyt b(5), this finding suggests that the cofactor is kinetically trapped under physiologically relevant conditions. This conclusion is confirmed by H-1 MMR studies which show that the hemin isomeric ratio changes when the protein is incubated for several hours at 68 degrees C. Interestingly, the orientational isomer favored in OM cyt b(5) is the form less favored in all Other known cytochromes b(5).