O₂- and H₂O₂-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXα complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O₂ or H₂O₂; 2) the verdoheme reactivity with O₂ is the lowest among the three O₂ reactions in the HO catalysis, and the newly found H₂O₂ pathway is ∼40-fold faster than the O₂-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O₂ or H₂O₂ to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp¹⁴⁰ in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O₂ and H ₂O₂ on the verdoheme iron with the aid of a nearby water molecule linked with Asp¹⁴⁰. These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H₂O₂-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.