Abstract

The nature of cytochrome P-420 present in "CO-binding particles" was studied. This problem was approached by investigating the transferability of heme from CO-binding particles, containing only P-420, obtained through the treatment of microsomal fractions with steapsin, to human serum albumin. In addition, the reconstitution of cytochrome P-420 from "heme-depleted" protein derived from CO-binding particles was investigated. Tritiumlabeled cytochrome P-450 was prepared by treatment of rats with [3,5-³H]δ-aminolevulinic acid. The heme of cytochrome P-450 was not transferred to albumin. However, up to 83% of the heme present in CO-binding particles was transferred to albumin after 40 min of incubation. The resulting heme-albumin complex was identified as ferrihemalbumin (methemalbumin) by the criteria of millimolar extinction coefficient, CO difference spectrum, and fractionation profile. CO-binding particles, from which heme had been removed by albumin, could be reconstituted by treatment with methemalbumin. The resulting CO-binding particles showed millimolar extinction coefficients and CO difference spectra indistinguishable from control CO-binding particles. These results suggest that the cytochrome P-420 present in CO-binding particles could represent several complexes of heme and heme-binding sites of a protein, preferentially that of denatured apoprotein of cytochrome P-450, rather than one structural entity. Finally, in regard to the nature of heme-apoprotein interactions in CO-binding particles, it appears that, in comparison to cytochrome P-450, the hemeapoprotein interaction in these particles is modified so that the heme moiety is exposed to the surroundings. Thus, in CO-binding particles, proteins with high heme-binding affinity can readily remove the heme from its apoprotein.