Alkyl Peroxides Reveal the Ring Opening Mechanism of Verdoheme Catalyzed by Heme Oxygenase

Abstract

Heme oxygenase (HO) catalyzes heme catabolism through three successive oxygenation steps where the substrate heme itself activates O2. Although a rate-determining step of the HO catalysis is considered as third oxygenation, the verdoheme degradation mechanism has been the least understood in the HO catalysis. In order to discriminate three possible pathways proposed for the verdoheme ring-opening, we have examined reactions of the verdoheme-HO-1 complex with alkyl peroxides, namely MeOOH. Under reducing conditions, the MeOOH reaction afforded two novel products whose absorption spectra are similar to but slightly different from that of biliverdin. HPLC, ESI-MS, and NMR analysis show that these products are 1- and 19-methoxy-deoxy-biliverdins. The addition of a methoxy group at one end of the linear tetrapyrrole unambiguously indicates transient formation of the Fe-OOMe intermediate and rearrangement of its terminal methoxy group to the alpha-pyrrole carbon. The corresponding OH transfer of the Fe-OOH species is highly probable in the H2O2-dependent verdoheme degradation and is likely to be the case in the O2-dependent reaction catalyzed by HO as well.

A New Way to Degrade Heme

“…Apparent absence of CO formation indicates that the MhuD catalysis does not involve verdoheme, which is the key intermediate of ring cleavage by HO. Although the ring opening mechanism of verdoheme has been elucidated in HO enzymes (6,(33)(34)(35), essentially no mechanistic information is available for ring cleavage not via the verdoheme species.…”

Reduction of oxaporphyrin ring of CO-bound α-verdoheme complexed with heme oxygenase-1 by NADPH-cytochrome P450 reductase

“…In the physiological degradation of verdoheme by HO, though conclusive evidence is still lacking, we assume that O 2 exerts a similar effect as CO on the redox state of oxaporphyrin ring of verdoheme. It is pertinent, and supported by circumstantial evidence [9,13,16,25,26], that the initial step of the verdoheme degradation is the binding of O 2 , followed by the reduction of the oxaporphyrin ring (path B in Fig. 1), whereby FAD can provide one electron directly to verdoheme in its complex with HO.…”

“…In fact, excess exogenous CO [9,25] and cyanide [16,25] bound to the verdoheme iron inhibit the degradation of verdoheme in the HO reaction. Furthermore, binding of O 2 and H 2 O 2 to the iron of ferrous verdoheme-rHO-1 complex has been indicated by stopped-flow experiments [13,26]. Thus, it is reasonable to consider that binding of O 2 to verdoheme iron occurs first, and that subsequent oneelectron reduction initiates verdoheme cleavage.…”

Crystallographic studies of heme oxygenase complexed with an unstable reaction intermediate, verdoheme

“…6). The product in this reaction with ROOH is not normal biliverdin, but alkoxy-biliverdin [60], which indicates that only the Fe-OOR pathway is possible. This finding strongly suggests that the Fe-OOH species would be generated when reacted with O 2 or H 2 O 2 .…”