Subunit selectivity in the monooxygenase-like activity of tetrameric hemoglobin.
The aniline hydroxylase activity of adult rabbit hemolysates (1 mM with respect to hemoglobin concentration) was found to be 80 pmoles of p-aminophenol formed per minute per milliliter. This value is comparable to that observed for adult human hemolysates. The characteristics of this O2-requiring aromatic p-hydroxylation reaction are typical of the monooxygenase reactions catalyzed by the liver microsomal cytochrome P-450 system. Both systems are inhibited by carbon monoxide, which coordinates directly with the heme iron atoms of the respective hemoproteins. With the use of 13C-NMR spectroscopy, separate, well-resolved signals were observed for 13C-enriched carbon monoxide bound to the alpha- and beta-subunits of the tetrameric (alpha 2 beta 2) rabbit hemoglobin. By appropriately adjusting conditions, the hemoglobin was converted into hybrids of ligation varying from full oxygenation to intermediate forms in which the oxygen was progressively replaced by 13CO, first on the beta-subunits, then on the alpha-subunits until full CO ligation was accomplished. The state of ligation of the hemoglobin in each case was determined from the integrated areas of the signals in the corresponding 13C-NMR spectra. The corresponding aniline hydroxylase activity of the rabbit hemolysates containing such hybrids revealed that the monooxygenase activity of intact tetrameric hemoglobin is determined predominantly (if not exclusively) by the ligation of the beta-subunits. To the best of our knowledge, this is the first report of differential subunit behavior for a monooxygenase-like enzymatic activity.
D000815:Aniline Hydroxylase / Q000378:metabolism; D000818:Animals; D002248:Carbon Monoxide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits
D006454:Hemoglobins; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D000815:Aniline Hydroxylase