The reactivity of α‐hydroxyhaem and verdohaem bound to haem oxygenase‐1 to dioxygen and sodium dithionite

Abstract

Recently we have shown that ferric α‐hydroxyhaem bound to haem oxygenase‐1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196–18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric α‐hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous α‐hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203–213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. α‐hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of α‐hydroxyhaem with haem oxygenase may be small and a substantial amount of free α‐hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air‐sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH‐cytochrome P450 reductase.

Electrochemical reduction of ferrous α-verdoheme in complex with heme oxygenase-1

“…Indeed, we previously observed a spectrum similar to that shown in Fig. 4A when the verdoheme-rHO-1 complex was reduced with Na 2 S 2 O 4 under anaerobic conditions and tentatively assigned this spectrum to a π-neutral radical of verdoheme [8].…”

“…The redox potential of the oxaporphyrin ring of the ferrous verdoheme-rHO-1 complex was far lower than those of both flavins of CPR, and of NADPH (Table 1). It should be noted that a verdoheme π-neutral radical-like spectrum does not appear in the NADPH/CPR-supported degradation of ferrous verdoheme complexed with rHO-1 [8]. Thus, we consider it very unlikely that CPR can catalyze the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme in complex with rHO-1.…”

“…Thus we judged that a π-neutral radical of ferrous verdoheme in complex with rHO-1 was generated by the electrochemical reduction. This redox reaction was only partially reversible (data not shown), probably due to a reaction leading to nonproductive decomposition of verdoheme, that does not lead to biliverdin [8]. While the immediate product of the reaction has yet to be characterized, a two-electron-reduced anion form of ferrous verdoheme has been indicated as a possible candidate [32].…”

“…1 [4,5]. The first step is the oxidation of heme to α-hydroxyheme, requiring O 2 and two electrons [6][7][8]. The second step, also requiring O 2 and an electron, is the formation of ferrous α-verdoheme from α-hydroxyheme with the concomitant release of hydroxylated α-meso carbon as CO [9][10][11][12].…”

“…This redox potential value suggests that verdoheme in complex with rat HO-1 (rHO-1) may be reduced by CPR, because the redox potential of FMN (semiquinone/reduced) in rat CPR is −0.270 vs NHE [23]. However, in the degradation of ferrous verdoheme complexed with rHO-1 in the NADPH/CPR system, a spectrum such as that of ferrous verdoheme π-neutral radical has never been observed [8]. On the other hand, the binding of O 2 to the ferrous iron of the verdoheme-rHO-1 complex has been hypothesized as an alternative mechanism for the HO reaction [5,15].…”