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

Abstract

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to α-hydroxyheme, α-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme π-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the oneelectron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be −0.47 ± 0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the π-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O 2 to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

“…4A) and represents a well-defined direct electrochemical response of a heme degrading enzyme. This E m value is similar to that for rat [−87 mV (35)] and human [−65 mV (36)] HO-1 measured by spectroelectrochemistry. Aerobic CV of IsdI (20 mV∕ sec scan rate) in the same buffer exhibited a substantial reduction wave at approximately þ50 mV (vs. SHE) (Fig.…”

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

“…The midpoint potential for the cationic/neutral couple of ferrous verdoheme in complex with rHO-1 in the presence of CO ligand is approximately 70 mV higher than that in the absence of exogenous ligand (−0.465 ± 0.003 V vs NHE) (Fig. S1, Table 1) [24].…”

“…The reconstitution and purification of α-verdoheme-rHO-1 complex were performed as described previously [11,24,29]. Unless otherwise stated, the following manipulations were carried out under anaerobic conditions in a UNILAB glove box system (MBRAUN, Garching, Germany) filled with nitrogen gas.…”

“…The fractions containing the ferrous verdoheme-rHO-1 complex were combined and concentrated by ultrafiltration with Microcon YM-10 membrane (Millipore). The ferrous verdoheme-rHO-1 complex in 0.1 M potassium phosphate buffer, pH 7.0, exhibited absorption maxima at 400 (ε = 50.2 mM −1 cm −1 ), 534 and 688 nm [11,13,24]. …”

“…The redox potential reported (−0.903 V vs Ag/Ag + electrode [22] or −0.269 V vs the normal hydrogen electrode (NHE)) suggested that the verdoheme complexed with HO-1 could be reduced by CPR, because it is close to the redox potential of FMN (semiquinone/reduced) in rat CPR, −0.270 vs NHE [23]. Recently we carried out an electrochemical titration of exogenous ligand-free ferrous verdoheme-rat HO-1 (rHO-1) complex and obtained a redox potential of −0.465 V vs NHE for the one-electron reduction of oxaporphyrin ring of verdoheme [24]. Thus we concluded that the one-electron reduction of the oxaporphyrin ring of the ferrous verdoheme-rHO-1 complex by CPR is unlikely to occur from considerations (see Fig.…”