Studies on the enzymatic mechanism of microsomal heme oxygenase were made utilizing various porphyrins and metalloporphyrins of different ring substituents and central metal ions. Co-heme (cobalt protoporphyrin IX) was a substrate for the enzyme and the product of its oxidative metabolism was identified as the natural bile pigment, biliverdin IX.alpha. isomer. Metalloporphyrins, which do not bind O2 (Ni, Mn and Sn protoporphyrin IX), were not substrates for heme oxygenase, although they could competitively inhibit oxidation of reactive substrates for the enzyme. The presence of lipophilic substituents on pyrrole rings I and II, as well as a central metal atom, were required for the heme oxidation reaction to occur. The oxidative cleavage of Co-heme displayed typical characteristics of an enzyme-mediated reaction, and the oxidation of this substrate, as well as that of Fe-heme (Fe protoporphyrin IX), could be supported with either NADPH or NADH. The enzyme and its substrate may form a transitory hemoprotein which can activate O2 for cleavage of the heme tetrapyrrole ring. In this formulation, heme as substrate for heme oxygenase is synonymous with heme as prosthetic group for the enzyme.