OXYGEN AND ONE REDUCING EQUIVALENT ARE BOTH REQUIRED FOR THE CONVERSION OF ALPHA-HYDROXYHEMIN TO VERDOHEME IN HEME OXYGENASE

Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-h eme oxygenase complex, which is the first intermediate in the catalyti c reaction. The active site structure of the complex was examined by o ptical absorption, EPR, and resonance Raman spectroscopies. In the fer ric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structur e of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and conseq uently the prosthetic group assumes a porphyrin structure. The alpha-h ydroxyheme group undergoes a redox-linked conversion between a keto an d an enol form. The ferric alpha-hydroxyheme reacts with molecular oxy gen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. React ion of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxyge n also yields the verdoheme-enzyme complex. We conclude that the catal ytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxyge nase requires molecular oxygen and one reducing equivalent.