The step of carbon monoxide liberation in the sequence of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system.

Oxidative degradation of protoheme to biliverdin IXa catalyzed by the microsomal heme oxygenase system proceeds in the following sequence: protoheme, a-hydroxyheme, the 688-nm substance, a biliverdin-iron complex, and biliverdin.In the heme oxygenase reaction, the a-methene carbon of heme is liberated as carbon monoxide.The 688-nm substance, a newly found intermediate (Yoshida, T., Noguchi, M., and Kikuchi G. (1980) J.Biochem (Tokyo) 88, 557-563), has an extremely high affinity for carbon monoxide and, therefore, when the heme-heme oxygenase complex was incubated with the NADPH-cytochrome P-450 reductase system under a gas mixture of air and CO at a volumetric ratio of 5:1, about 80% of the heme was found to stay at the level of the CO-reduced form of the 688-nm substance.Taking advantage of these properties of the 688-nm substance, an attempt was made to determine whether carbon monoxide is liberated before or after the step of the 688-nm substance, by using 14C-labeled heme synthesued from [5-'4C]G-aminolevulinic acid.The complex of 14C-heme and heme oxygenase was incubated with the NADPH-cytochrome P-450 reductase system in either air or a gas mixture of air and CO, and the content of 14C0 in the gas phase was analyzed by means of converting 14C0 into 14C02 by oxidation.In either reaction system, practically equal amounts of 14C02 were obtained; the radioactivities recovered in COz were 11 to 12% of the original radioactivity of 14C-heme used.The values of 11 to 12% are very close to the theoretical value of 12.5% (or one-eighth) which is expected to be recovered in C 0 2 when all the I4C-heme was completely degraded to biliverdin.These findings clearly indicate that carbon monoxide is liberated at the step of the formation of 688-nm substance from a-hydroxyheme and, hence, the 688-nm substance evidently lacks the a-methene carbon.