230

ORIGINAL
Electrons released from both flavins of NADPH-P450 reductase
contribute to the reductive mobilization of iron from ferritin
Kazumi Takaishia and Hiroshi Kitahatabab

Department of Dental Anesthesiology, Tokushima University Hospital, Tokushima, Japan
Department of Dental Anesthesiology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan

Abstract : Ferritin, an iron storage protein, plays an important role in iron homeostasis. The mechanism ofreductive mobilization of iron from ferritin has not been clarified yet despite many studies. The aim of this study
was to assess the mechanisms of the mobilization of iron from ferritin by NADPH P-450 reductase. Nucleotidedependent flavoenzymes generated significant mobilization of iron from ferritin. The possibility of reductive
mobilization of iron from ferritin by electrons released from flavin sites or heme site of two flavoenzymes wasinvestigated to elucidate the mediator-independent mechanisms of such reductive mobilization. The mobilization
by NADPH-P450 reductase in the presence of ferricyanide increased threefold, while in the presence of cytochrome C increased thirteen-fold. These results indicate that electrons released from both flavins of NADPH-P450
reductase contribute to the reductive mobilization of iron from ferritin. The mechanism of the mobilization ofiron from ferritin is discussed. J. Med. Invest. 66 : 230-232, August, 2019
Keywords : Ferritin, Iron, Electron, Flavoenzyme, Reductive mobilization

INTRODUCTION

MATERIALS AND METHODSFerritin is an iron storage protein in many mammalian cells(1). Cellular iron homeostasis is maintained by regulation of theexpression of ferritin and the transferrin receptor.
The ferritin molecule consists of a mineral core of hydrated
ferric oxide and 24 protein shells, and the iron content in the core
consists of a maximum of 4500 atoms. Two types of channels, six
four-fold hydrophobic channels and eight three-fold hydrophilicchannels, are generated by assembly of the protein shells. The
hydrophobic channel is 0.3-0.4 nm wide and 1.2 nm long, andthe hydrophilic channel is 0.3-0.4 nm wide and 0.5 nm long, butis blocked by metal irons (2, 3). The route of iron deposition andmobilization via the hydrophobic channel has been the subject ofinvestigations. The iron in the core can be mobilized from ferritin byreduction of ferric iron, and iron mobilization has been observedon the addition of biological reductants such as dihydroflavin(4-7), dihydrolipoate (7, 8), superoxide anion radical (9, 10), andnitric oxide (11). In recent studies, it was shown that the iron release from ferritin occurred through lysosomal proteolysis or Nuclear Receptor Coactivator-4 – mediated ferritinophagy (12, 13).
However, this process is relatively slow from the perspective of
rapid reactivity of iron ion and is related to degradation of ferritin
cage (12, 14). It has been suggested that the reductant must penetrate into the interior of the channel in order to reduce the iron.
Whether ferritin iron reduction involves direct access by thereductant into the central cavity of the molecule or electron
tunneling via the hydrophobic channel has remained unclear.
The purpose of our study was to investigate the molecular mechanisms of the mobilization of iron from ferritin by the nucleotide-dependent flavoenzymes.

Ferritin (horse spleen) and NADPH-P 450 reductase (rabbitliver) were obtained from Sigma-Aldrich Co. (St. Louis, MO).
The standard reaction mixture for iron mobilization fromferritin contains 1.9 μM ferritin, 1U NADPH-P 450 reductase,
20 μM bathophenanthroline disulfonic acid 2Na salt in 20 mM
phosphate buffer, pH 7.4. Bathophenanthroline is used as aquantity regent of iron by binding to ferrous iron and forming
orange-red chelate. We measured the quantity of released ferrous iron in real time using the spectrophotometer (U-3900,Hitachi High-Tsch Science Corporation, Japan) after adding 100
μM NADPH to the standard reaction mixture. The quantity ofreleased ferrous iron was calculated from absorbance at 530 nmby using a molar absorbance coefficient of 22.1 × 103 M-1 cm-1 forbathophenanthroline.
To investigate the effect of superoxide dismutase on the reductive mobilization of iron, 10 μM superoxide dismutase added tothe standard reaction mixture. In another experiment to elucidate the mechanism of ferritin iron reduction, 2 mM ferricyanide
or 6.7 μM cytochrome C was added each to the standard reaction
mixture.
No less than 4 experiments were performed separately. The
results are expressed as the representative data for all theexperiments.

Received for publication February 8, 2018 ; accepted February 2, 2019.
Address correspondence and reprint requests to Kazumi Takaishi,
Department of Dental Anesthesiology, Tokushima University
Hospital, 3-18-15 Kuramoto, Tokushima 770-8504, Japan andFax : +81-88-633-7368.

RESULTS

Mobilization of iron from ferritin by NADPH-P 450 reductase
NADPH-P 450 reductase mobilized iron from ferritin asshown in Fig.1. This reductive mobilization was reduced by 6%
in the presence of superoxide dismutase (Table 1). It was not
significantly reduced in the presence of superoxide dismutase,indicating that the superoxide anion radical did not mediate thereduction of ferritin iron.

The Journal of Medical Investigation Vol. 66 2019

The Journal of Medical Investigation Vol. 66 August 2019

Fig 1. Time course of iron mobilization from ferritin by means ofNADPH P-450 reductase.
The reaction mixture contains 1.9μM ferritin, 1U NADPH-P 450 reductase, 100μM NADPH, 20μM bathophenanthroline disulfonic acid
2Na salt in 20mM phosphate buffer, pH 7.4. The quantity of released
ferrous iron was calculated from absorbance at 530 nm by using a-3-1-1molar absorbance coefficient of 22.1 × 10 M cm for bathophenanthroline.

Table 1 Effects of Superoxide dismutase on iron mobilization fromferritin by NADPH P-450 reductase

Rate of iron mobilization (Fe2+ μM / 10min)
Control

2.71

Superoxide dismutase (10 μM)

2.55

The effects of ferricyanide and cytochrome C on the mobilization ofiron from ferritin by NADPH-P 450 reductase
We performed another study to elucidate the mechanism offerritin iron reduction. It is recognized that the external electron
acceptors of FAD and FMN of diflavin reductase family are
ferricyanide and cytochrome C, respectively. Here, the reductive
mobilization of iron from ferritin is investigated in the presenceof external electron acceptors. The mobilization in the presenceof ferricyanide increased threefold, while in the presence of cytochrome C increased thirteen-fold as shown in Table 2.

Table 2 Effects of ferricyanide and Cytochrome C on iron mobilization from ferritin by NADPH-P450 reductase

Rate of iron mobilization (Fe2+ μM / 10min)
Control

2.71

Ferricyanide (2 mM)

8.58

Cytochrome C (6.7 μM)

34.29

DISCUSSION
In this study, NADPH-P450 reductase was found to be able tocatalyze the mobilization of iron from ferritin. It was reportedthat the reductive mobilization of iron from ferritin is dependent
on the relative rate of NADH oxidations, dissolved O2 consumption and mineral core reduction in the study using recombinantfrog M ferritin (15). It was also reported that the iron mobilization from ferritin is dependent on the concentration of dissolved
oxygen in solution (16). However, Johnson LE et al. reported that231

the reductive mobilization of iron by reduced flavin mononucleotide (FMNH2) was limited by the concentration of FMNH2
under their experimental conditions excluded the possibility ofoxygen diffusion into the reaction mixture, and that iron reductive mobilization was independent on the presence of chaotropes(17). In our experiment, the influence of oxygen diffusion on ironrelease was extremely limited because the iron mobilization wasreduced by only 6% in the presence of superoxide dismutase.
Moreover, Johnson LE et al. showed that the diffusion of FMNH2
through the ferritin pores was unlikely mechanism by the experiments about encapsulation of FMN inside the ferritin cavity(17).
A further investigation is needed to clarify the mechanismof ferritin iron reduction by the nucleotide-dependent flavoenzymes. One possibility is thought to be that the electron fromeither enzyme could gain direct access into the core through theferritin channel and thus activate the reduction of ferritin iron.
NADPH-P 450 reductase exerts their ferricyanide reductive
activity by means of electrons from the FAD site and the cytochrome C reductive activity by means of electrons from the FMN
site (18, 19). In our experimental conditions, the reductive mobilization of iron from ferritin is about three folds by the additionof ferricyanide. On the other hand, the reductive mobilization ofiron from ferritin is about thirteen folds by the addition of cytochrome C, indicating that electrons released from both flavins of
NADPH-P450 reductase contribute to the reductive mobilizationof iron from ferritin.
The hop distance of electron transfer from site to site of themetal complex model is 1-2 nm. The rate constant is 8 × 10 sec
at a distance of 1.13 nm and drops during metal ion separation
by 10 seconds for each 0.3 nm increment (20). While it can beassumed that a hop distance of 2 nm is close to the limit of usefulness, it can occur in proteins over a distance of more than 1nm (21).
Access to the interior of the shell of the ferritin molecule ispossible only via a channel. There are two types of channels
through which small molecules can gain access to the central
core of the molecule. The hydrophobic channel is 0.3-0.4 nm indiameter and 1.2 nm long, while the hydrophilic channel is 0.30.4 nm in diameter and 0.5 nm long (2, 3). In theory, therefore,
the electrons could gain direct access via one of these channels to
the central core of the ferritin molecule.
Ferritin iron cores can be reduced by one electron per ironaccompanied by an uptake of two protons per electron from thesurrounding medium (22). The midpoint potential of ferritin
iron is E = − 190mV (23). The midpoint potential of NADPH-P
450 reductase is E = − 274 mV (ox/sq), E = − 371 mV (sq/hq) for
FAD and E = − 109 mV (ox/sq), E = − 279 mV (sq/hq) for FMN(24). Since the midpoint potential for FAD of NADPH-P 450
reductase is much lower than the midpoint potential of ferritin
iron, flavin semiquinone of the enzyme is provided thermodynamically for the mechanism of electron transfer to ferritin iron.
For these reasons, the reductive mobilization of iron from ferritin
may be induced by direct electron transfer from the flavin site ofNADPH-P 450 reductase to the ferritin core.
Iron homeostasis is related to a variety of important physiological functions (25). A large quantity of free iron released from ferritin generates highly reactive hydroxyl radical from superoxideand hydrogen peroxide. It can lead the control of the pathophysiology related to free radicals to explore the mechanism of ironrelease from ferritin. It is mandatory to investigate more about
the mobilization of iron from ferritin.

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K. Takaishi and H Kitahata Reductive mobilization of iron by electrons

CONCLUSION
The iron mobilization by NADPH-P450 reductase from ferritin in the presence of ferricyanide or cytochrome C increased
markedly. It is suggested that electrons released from bothflavins of NADPH-P450 reductase contribute to the reductive
mobilization of iron from ferritin.

CONFLICT OF INTEREST
None of the authors have any conflicts of interest to declare.

ACKNOWLEDGEMENT
We are greatly indebted and wish to express our profoundgratitude to Prof. Emeritus Aki K for his advice and direct guidance throughout the entire course of this study.
This study was supported in part by a Gran-in-Aid for Scientific Research from Japan Society for the Promotion of Science(#23792350).

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