Reaction of Monomeric and Dimeric Species of Heme a with Imidazole and Cyanide

Heme a, the prosthetic group of cytochrome oxidase [EC 1.9.3.1], was extracted from bovine heart muscle and purified by chromatography on a DEAE-cellulose column. The relationship between the absorption spectra and state of aggregation of heme a in solution was investigated. Heme a was found to be polymeric in a phosphate buffer solution, and converted to the dimeric form by non-ionic detergents. The reaction of heme a with imidazole and HCN was studied in the anionic (sodium dodecyl sulfate), non-ionic (Triton X-100), and cationic (cetyltrimethylammonium bromide) solutions. Heme a reacted with imidazole to form a di-imidazole complex, but the reactivities were different depending on the ionic nature of the detergents used; heme a reacted with imidazole more easily in the anionic detergent solution than in the cationic. HCN reacted not only with the heme iron but also with the formyl group of heme a to form cyanhydrin in the neutral pH region, but not on the alkaline side. For induction of a spectral change of the same extent, a higher concentration of HCN was required in the anionic detergent solution than in the cationic. The reaction of the formyl group of heme a with HCN was investigated in detail in a Triton X-100 solution containing imidazole. It was established that the reactivity of the formyl group was dependent on the redox state of the heme iron; it was more reactive in the oxidized form than in the reduced.