Features of the reaction of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system.

Abstract

The heme oxygenase system was reconstituted from a heme oxygenase preparation highly purified from pig spleen microsomes and a partially purified NADPHcytochrome c reductase from pig liver microsomes. In the reconstituted heme oxygenase reaction, the relationship between the rate of heme degradation and the heme concentration was sigmoidal, and the heme concentration which gave the half-maximum rate of the reaction was about 1 PM. The kinetic behavior of the heme oxygenase reaction was not appreciably affected by the addition of bovine serum albumin, but when added with human serum albumin which easily binds with heme to form methemalbumin, the sigmoidal nature of the activity-substrate relationship was considerably pronounced and a higher heme concentration was required for the half-maximum rate ofthe reaction. Free heme should be the substrate of the heme oxygenase reaction. The complex of ferric heme and heme oxygenase was prepared by using a substantial amount of the purified heme oxygenase preparation, and the complex was incubated with NADPH and NADPH-cytochrome c reductase. By this reaction, the heme bound to heme oxygenase was easily and quantitatively converted to biliverdin. The heme bound to heme oxygenase also rapidly decomposed when the complex was incubated with ascorbic acid. The final product in the latter reaction system, however, was not biliverdin but appeared to be a biliverdin l iron complex which probably remained attached to the heme oxygenase protein. The final product in the reaction system with ascorbic acid was converted to biliverdin when NADPH and NADPHcytochrome c reductase were added, suggesting that reduction of iron may be required for the release of iron from the biliverdin*iron complex. The final product in the ascorbate system also yielded biliverdin when desferrioxamine was added. Moreover, the rate of heme degradation in the reconstituted heme oxygenase reaction was increased considerably by the addition of desferrioxamine. The release of iron may be an important step in determining the rate of the overall heme oxygenase reaction. The heme oxygenase reaction was not inhibited by various scavengers of singlet oxygen, superoxide anion, and hydroxyl radical. Probably the molecular oxygen which is activated through binding with the heme on the heme oxygenase protein may be directly utilized