Abstract
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.
Highlights
Biochem (Tokyo)88, 557-563), has an extremely high affinity for carbonmonoxide and, when the heme-heme oxygenase complexwas incubated with the NADPH-cytochromeP-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 tlheevel of the CO-reduced form of the 688-nm substance
Taking advantage of these propertiesof 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 acidT. he complex of heme degradation in the heme oxygenase reaction would proceed in the following way
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 2when all the I4C-hemewas completely degraded to biliverdin. These findings clearly indicate that carbon monoxide is liberated at the step of the formation of688-nm substance from a-hydroxyheme and, the 688-nm substance lacks the a-methene carbon
Summary
The reaction mixture placed in the main compartment of the flask contained, in a final volunle of Reagents-The following reagents were purchased from the 1 ml, 6 nmol of the "C-heme-heme oxygenase complex (5280 dpm), sources indicated [5-14C]ALA1 hydrochloricacid (47.5 Ci/mol), from 0.1 unit of NADPH-cytochrome P-450 reductase, and 110 mM pots>. 10-24 mesh) from Nakarai Chemical Co., Kyoto; ,&acetylphenylhy- flask in ice water. Other chemicals were obtained from commercial sources described a glass tube (0.8 X 10 cm) filled with Hopcalite I1 and attached with previously [2]. Preparation of I4C-labeled Heme-I4C-heme was synthesized by to a counting vial and 10 ml of toluene scintillation mixture [23] were incubating [5-I4C]ALAwith a rabbit reticulocyte lysate according to added to itfor the determinationof radioactivity. The crystalline hemewas further purified according to the methodof Healey zt a/. [19] to eliminate possible contaminants of high radio-
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