Characterization of Enzyme-like activity of human hemoglobin. Properties of the hemoglobin-P-450 reductase-coupled aniline hydroxylase system.
Human hemoglobin was characterized as an enzyme in a reconstituted aniline hydroxylase system containing hemoglobin, NADPH, rat liver cytochrome P-450 reductase, aniline and atmospheric O2. This system catalyzed p-aminophenol formation (turnover number 0.2 mol/min/mol of hemoglobin) with an efficiency similar to that which has been reported for either microsomal cytochrome P-450 or cytochrome P-450 solubilized from rat liver. The rate of the reaction was linearly dependent on hemoglobin concentration up to approximately 1 nmol of hemoglobin/ml. This linear range of hemoenzyme concentration is also similar to cytochrome P-450-catalyzed reactions. Unlike the cytochrome P-450 system, the hemoglobin system did not require a lipid cofactor for maximal activity, and much less reductase was needed for maximal activity. Aniline displayed typical Michaelis-Menten saturation kinetics as substrate, and its Km (8 mM) was the same in the absence of presence of the reductase. Catalase essentially completely inhibited p-aminophenol formation in the absence or presence of reductase. In contrast, superoxide dismutase inhibited the reductase-mediated reaction only to a small extent (if at all). No detectable hydrogen peroxide accumulated during the course of the reaction in the absence of catalase. These findings suggested a hypothetical mechanism for hemoglobin-catalyzed hydroxylation of aniline involving a hemoglobin-bound form of hydrogen peroxide (aniline-Hb3+-OOH-) as an intermediate preceding the rate-determining formation of products.
D000815:Aniline Hydroxylase / Q000097:blood*; D002374:Catalase / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000097:blood*; D003579:Cytochrome Reductases / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008706:Methemoglobin / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000097:blood*; D013482:Superoxide Dismutase / Q000494:pharmacology
D006454:Hemoglobins; D008706:Methemoglobin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D005949:Glucose Oxidase; D002374:Catalase; D000815:Aniline Hydroxylase; D013482:Superoxide Dismutase; D003579:Cytochrome Reductases