Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation

Article abstract of DOI:10.1021/acs.biochem.8b01314

Oxyhemoglobin (HbO₂) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO₂ to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO₂ coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H₂O₂, NO, and NaNO₂) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO₂, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO₂ is the same as those of IHP and DPG. ³¹P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO₂. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO₂ with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO₂ coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO₂.

Full text of DOI:10.1021/acs.biochem.8b01314

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Article
Antioxidative pseudo-enzymatic mechanism of NAD(P)H coexisting
with oxyhemoglobin for suppressed methemoglobin formation
Magohei Yamada, Takashi Matsuhira, Keizo Yamamoto, and Hiromi Sakai
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01314 • Publication Date (Web): 21 Feb 2019
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Revised manuscript submitted to Biochemistry
ID: bi-2018-013145-R1
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Antioxidative pseudo-enzymatic mechanism of NAD(P)H coexisting with
oxyhemoglobin for suppressed methemoglobin formation
Magohei Yamada, Takashi Matsuhira, Keizo Yamamoto, and Hiromi Sakai*
Department of Chemistry, Nara Medical University
840 Shijo-cho, Kashihara, Nara 634-8521, Japan
* To whom correspondence should be addressed:
Prof. Hiromi Sakai, Department of Chemistry, Nara Medical University
840 Shijo-cho, Kashihara, Nara 634-8521, Japan
Tel. and Fax: +81-744-29-8810; E-mail: hirosakai@naramed-u.ac.jp
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Abstract
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Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and
oxidants-induced metHb formation based on the pseudo-catalase (CAT) and pseudo-superoxide
dismutase (SOD) activities. In this work we compared the effects of NADH with those of
NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative
mechanisms. Results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting
with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (< 40
min) facilitated by oxidants (H2O2, NO, NaNO2) was hindered by NADPH. These effects were
similar to those of NADH. However, we found that NADPH is thermally unstable compared to
NADH, and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to
metHb such as 24 h. Lineweaver–Burk plots clarified that the Michaelis constants of these
pseudo-enzymatic activities are of millimolar order. Addition of inositol hexaphosphate (IHP)
and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also
weakly with HbO2, showed competitive inhibition of pseudo-enzymatic activities. These results
suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and
DPG. 31P NMR surely clarified 1:1 stoichiometric NADH binding to HbO2. HPLC analysis
showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations
predicted that the binding site of the relax-state HbO2 with NAD(P)H is equal to those with IHP
and DPG. Collectively, the pseudo-enzymatic activities of HbO2 coexisting with NAD(P)H is
induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.
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(250 words)
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INTRODUCTION
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Ferrous oxyhemoglobin (HbO2) gradually autoxidizes to form ferric methemoglobin
(metHb) that cannot bind oxygen.1,2 During this process, superoxide anion (O2·–) is released
(HbO2 → metHb + O2·–), forming hydrogen peroxide3 via a disproportionation reaction,
enhancing further oxidation of HbO2 and deoxyHb to converting them to metHb. In addition to
these reactive oxygen species (ROS), reactive nitrogen species (RNS) such as nitric oxide (NO)
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–
and nitrite (NO2 ) present in the blood contribute to conversion of Hb to metHb.4 Red blood cells
(RBCs) have various functions to reduce metHb and contain catalase (CAT) and superoxide
dismutase (SOD) decomposing ROS. Ferric metHb is reduced enzymatically to a ferrous state by
the action of NADH-cytochrome b5 reductase, which requires a substrate of reduced form of
nicotinamide adenine dinucleotide (NADH) as well as cytochrome b5 as an intermediate electron
mediator.5 Nicotinamide adenine dinucleotide phosphate (NADPH)-metHb reductase and
NADPH-flavin reductase, which require a coenzyme of NADPH, reduce metHb.6 The oxidized
form NADP+ is reduced to NADPH7 via the pentose phosphate pathway.
Our earlier report described that the coexistence of equimolar amount of NADH with
HbO2, with no corresponding enzymes described above, retarded autoxidation of HbO2 for 24 h
in a physiological condition (37 °C, pH 7.4).8 However, the similar molecule of NADPH did not
inhibit metHb formation. We reported that NADH coexisting with HbO2 showed pseudo-CAT
and pseudo-SOD activities.8 By trying co-encapsulation of NADH to artificial red blood cells
(liposome-encapsulated Hbs), we found that autoxidation of the artificial red blood cells was
inhibited in a rat model. In that experiment, the intra-liposomal NADH concentration was 1.5
mM: approximately 100 times greater than that in a human RBC (NAD+, 0.065 mM; NADH,
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0.00028 mM; 0.06528 mM in total).9 Actually, NADH is useful with artificial red blood cells for
clinical use because the safety of NADH is proven.10
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Our earlier study produced no detailed explanation of the mechanism of the pseudo-
enzymatic activities.8 Although NADPH is quite similar to NADH in terms of its structure and
standard redox potential, NADPH showed inferior inhibition effects on autoxidation of HbO2. As
described in this paper, we first investigated their pseudo-CAT and pseudo-SOD activities of
NAD(P)H coexisting with HbO2, their inhibitory effects on metHb formation induced by the
addition of oxidants (H2O2, NO, and NaNO2), and their thermal stability to clarify reasons for
similarities and differences between NADH and NADPH. Secondly, we strove to clarify the
mechanism of inhibition effects of metHb formation from the perspective of binding interaction
between HbO2 and NAD(P)H. The Michaelis constant of each pseudo-enzymatic activity was
determined. Inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG) are known to bind
weakly to HbO2 in a relaxed state.11,12 Profiles of the inhibition effect of IHP and DPG were
investigated. 31P NMR and docking simulations were conducted for the investigation of binding
sites of NAD(P)H on HbO2. The results provide insight and information related to the
antioxidative pseudo-enzymatic mechanism of NAD(P)H coexisting with HbO2.
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MATERIALS AND METHODS
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Purification of Hb from Red Blood Cells—Experiments using human-derived Hb were
approved by the ethical committee of Nara Medical University. Hb (a main component, adult
human hemoglobin) was purified from RBCs as described in earlier reports.13,14 One Hb is
consisted of two alpha subunits and two beta subunits. UniProtKB entry P69905 is for
hemoglobin subunit alpha, and entry P68871 is for beta. Outdated packed RBCs were provided
by the Japanese Red Cross Society. Purified HbO2 solution was obtained via carbonylation,
pasteurization, nanofiltration, dialysis, and decarbonylation.8 Absence of residual enzymatic
activities in the HbO2 solution was confirmed. MetHb was prepared using a method described in
an earlier report.8,15
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MetHb Formation Induced by Oxidants and Inhibitory Effect of NAD(P)H—NADH
and NADPH were purchased from Oriental Yeast Co., Ltd. and Sigma-Aldrich Corp.,
respectively. Three oxidants that promote metHb formation were used: (1) 1-hydroxy-2-oxo-3-
(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7; Dojindo Molecular Technologies Inc.),
(2) NaNO2 (Wako Pure Chemical Industries Ltd.), and (3) 30 wt% H2O2 (Wako Pure Chemical
Industries Ltd.). The HbO2 solution (30 μL, 10.1 g/dL) was diluted with 3 mL of PBS (pH 7.4;
Gibco Life Technologies Co.) (final Hb concentration, 100 mg/dL, 15.6 μM). Then it was
incubated with each of the oxidants at the molar equivalent to that of HbO2 at 37 °C. The metHb-
specific absorbance at 630 nm was measured to evaluate the effects of equimolar NAD(P)H on
metHb formation.
Thermal Stability Evaluation of NAD(P)H—NADPH and NADH (1.6 mM) were
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dissolved in PBS (pH 7.4). Then 10 mL of the solution was sealed in glass vials. Each vial was
incubated either for 7 days at 25 °C, 3 days at 30 °C, 2 days at 37 °C, or 3 h at 50 °C. During the
incubation NAD(P)H was oxidized by the dissolved dioxygen to form NAD(P)+. The change of
the concentration of the reduced form was monitored by the absorbance at 340 nm using a
spectrophotometer (V-660; Jasco Corp.) equipped with an integral sphere unit (ISV-722). After
each incubation condition, the percentage of the remaining reduced form (recovery) was
calculated from the decrease in the absorption at 340 nm. The time necessary to decrease to 80%
recovery (t0.8) and 20% recovery (t0.2) were calculated. Rate constant (k) for the oxidation of
NAD(P)H was calculated for each incubation condition. Graphs of Arrhenius plots were made to
obtain the activation energy (Ea).
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Pseudo-enzymatic Activities of NADPH–Hb Mixed System—Pseudo-CAT and pseudo-
SOD activity values were measured using a method reported earlier.8 (1) CAT activity was
measured in 1000-times diluted Hb solution based on the change in 230 nm absorbance of
H2O2.16 (2) SOD activity was measured in 100-times diluted Hb solution using pyrogallol
autoxidation method.17,18 Pyrogallol (Wako Pure Chemical Industries Ltd.) autoxidizes and
thereby generates superoxide. Then the solution becomes yellow with 420 nm absorbance. This
reaction is inhibited by the presence of SOD. The SOD activity was measured based on the
inhibitory effect. The analytes included purified HbO2, HbCO, and metHb solutions with or
without NADPH. The results were compared with previously reported values of NADH-Hb
mixed system and stroma-free hemolysates (SFHL) containing native SOD and CAT prepared
from blood of three individuals.
Kinetic Parameters of the Pseudo-enzymatic Activities and Influence of IHP and
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DPG—Lineweaver–Burk plots were made by plotting the inversed pseudo-enzymatic activities
(1/V) and the inversed concentration of NAD(P)H ([HbO2] = 1.5 mM, [NAD(P)H] = 1–15 mM).
The maximum enzymatic activity (Vmax) and the Michaelis constant (Km) were calculated from
the graphs. IHP and DPG were selected as potential inhibitors for pseudo-enzymatic activities
because they are known to bind HbO2. Both IHP and DPG were purchased from Sigma-Aldrich
Corp. The concentration of the potential inhibitor was equal to NAD(P)H. The values of Vmax, Km
and the inhibition dissociation constant (Ki) were calculated from Lineweaver–Burk plot data.
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Influences of the Addition of NAD+, NADP+, and PLP—For detailed insight into the
mechanisms, the oxidized forms of NAD(P)H, NAD+ and NADP+, were tested to ascertain
whether they show inhibitory effects. Pyridoxal 5'-phosphate (PLP) was also tested for the
potential inhibitor because PLP is known to bind to Hb as an allosteric effector. NAD+ and PLP
were purchased from Sigma-Aldrich Corp.; NADP+ was from Oriental Yeast Co. Ltd. (Tokyo,
Japan). Lineweaver–Burk plots were made by plotting the inversed pseudo-enzymatic activities
and the inversed concentration of NAD(P)H in the presence of equimolar NAD+, NADP+, or
PLP.
HPLC Analyses of the Autoxidation Intermediate Hybrids of HbO2 in the Presence of
NADH—The HbO2 solutions (1.5 mM) in PBS in the presence and absence of 1.5 mM NADH
were incubated at 37 °C. Aliquots of the solutions were periodically pipetted out and analyzed by
HPLC (Chromaster; Hitachi High-Technologies Corp., Tokyo, Japan) equipped with an ion-
exchanging column, TSKgel SP-NPR (Tosoh Corp, Tokyo, Japan). An analyte solution was
injected into the column through a sampler. Intact HbO2 (α2β2), partially oxidized intermediate
+
+
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hybrids (α2β2 , α2 β2), and totally oxidized metHb (α2 β2 ) were separated by the column using a
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5 min linear gradient from 0.05 to 0.2 M sodium chloride in 20 mM bis-Tris-HCl buffer ( pH
6.0 ) at a flow-rate of 1.5 mL/min. Effluent was monitored using a diode array detector (5430
Diode Array Detector; Hitachi High-Technologies Corp.) with absorbance at 411 nm, which
corresponds to the isosbestic point of metHb and HbO2. The assignments of the two intermediate
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+
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hybrid signals, α2β2 and α2 β2, and the totally oxidized α2 β2 were taken from Tomoda et al.27
31P NMR of NADH in the Presence of HbO2—The 31P-NMR with 1H decoupling
spectra of NADH were obtained on the JEOL JNM-ECZ500R (JEOL RESONANCE Co., Tokyo,
Japan), equipped with a 5 mm ROYAL probe, at 202.27 MHz. Chemical shifts were referenced
to 85% H3PO4 (0 ppm). The spectra were the results of accumulating 512 scans at 25.3 °C, with
32k data points, at a 45° pulse angle, 4 s relaxation delay, and 5.29 s repetition time. The analyte
solutions of NADH (1.5 mM) in PBS in the presence of HbO2 (0, 0.5, 1.0, and 1.5 mM) were
measured. The assignments of the chemical shifts were taken from the references.19-21 From the
integrated intensities of the signals, the ratio of the amount of NADH being interacted with HbO2
was calculated for all the analytes. We did not measure NADHP because of its instability that
might cause decomposition during the preparation of the analyte solutions and a long-term NMR
analysis.
Docking Simulation of NAD(P)H and Hb—All docking simulations were performed
using the MF myPresto v3.2 mmMPApp2 applications (FiatLux Co. Ltd., Tokyo, Japan). The
PDB files of the registered structure were converted into files for MF myPessto. The PDB files
used were HbO2 (PDB code, 1HHO22), deoxyHb (2HHB23), NADH (2JHF24), and NADPH
(1US025). Topology “Tplgene” data were made for “protein” (HbO2 or deoxyHb) and “ligand”
(NAD(P)H) for docking simulation. After the docking simulation, the main interatomic distances
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were measured using WebLab Viewer Lite – Molecular Visualization Software (WLViewerLite
40) with the data of the docking simulation. The figures of the binding sites were described using
the PyMOL Molecular Graphic System (Ver. 1.6, Schrödinger, LLC, New York).
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RESULTS
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MetHb Formation in HbO2 Solution by the Addition of Oxidants and Inhibitory
Effects of NADPH—According to our earlier study,8 HbO2 solution was purified via
carbonylation, pasteurization, nanofiltration, and decarboxylation. In the HbO2 solution, no other
protein was detected using high performance liquid chromatography, sodium dodecyl sulfate
polyacrylamide gel electrophoresis, or isoelectric focusing. Furthermore, no enzymatic activity of
SOD, CAT, NADH-metHb reductase, or NADPH-diaphorase was found, indicating complete
elimination of enzymes during purification.8 All experiments described below used this purified
HbO2 solution.
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The effects of addition of oxidants including H2O2, NaNO2, and NO to an HbO2 solution
were evaluated. For NO production, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-
triazene (NOC7) was used. It discomposes in aqueous solution generating NO. The addition of
oxidants induced metHb formation. HbO2 was oxidized almost completely in 40 min (Figure 1).
By contrast, the coexistence of NADPH and NADH caused clear retardation of metHb formation.
In the HbO2 solution with equivalent molar of NADPH, the levels of metHb after 40 min
incubation with H2O2, NaNO2, and NOC7 were 58%, 47%, and 63%, respectively of the levels
obtained in cases without NADPH. In fact, NADH showed 5–8% lower levels of metHb than
NADPH did, but the inhibitory effects were similar.
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Figure 1. Time course of metHb level (%) of HbO2 solution with and without NADPH (or
NADH) after the addition of oxidants: (A) H2O2, (B) NaNO2, and (c) NOC7. The initial
concentrations of HbO2, NADPH (or NADH), NaNO2, H2O2, and NOC7 were all 15.6 μM in the
cuvettes. The concentration of metHb (%) was calculated based on the increase of absorbance at
630 nm. The absorbance changes of Hb without NADPH (or NADH) after incubation at 25 °C
for 40 min were defined as 100%.
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Measurement of Pseudo-Enzymatic Activities of NADPH and Hb Solution Mixed
System—Table 1 presents pseudo-enzymatic activities (CAT and SOD) of NAD(P)H coexisting
with Hb. Data for NADH reported previously8 were inserted for comparison. Pseudo-enzymatic
activities for NADPH coexisting with HbO2 showed similar levels to those compared with those
for NADH (Entries No. 1–5). Combination with HbO2 showed higher activities compared to
those with metHb and HbCO (Entries No. 6–7). NADPH alone showed pseudo-activity, as did
NADH, based on the measurement principles (Entries No. 8–11), but they were much lower than
those of the mixture of NADPH and HbO2. Because both NADH and NADPH are known to react
with H2O2,26 increasing their concentrations showed higher pseudo-CAT activities based on the
measurement principles. The pseudo-SOD activities were low. Moreover, there was apparently
no concentration dependence. NAD(P)H coexisting with HbO2 showed comparable enzymatic
activities with those of stroma-free hemolysate (SFHL) containing native enzymes (Entry No.
12).
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Table 1. Pseudo-catalase (CAT) and pseudo-superoxide dismutase (SOD) activities in Hb solution
with or without coexistence of NADPH (and NADH as a reference8) a
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Entry State of Hb
No.
Hb
Potential
Potential
Antioxidant
concentration
(mM)
0.5
Pseudo-CAT
Pseudo-SOD
(×102 U/g Hb)
(mM)
Antioxidant
(×104IU/g Hb)
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NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
NADPH
(NADH)
5.2±1.1
(6.2±1.7)
17.6±2.1
(19.2±1.4)
23.6±1.6
(34.3±1.2)
26.8±0.8
(35.2±1.0)
36.0±2.1
(46.8±1.2)
4.2±1.0
1.5±1.4
(2.1±1.7)
6.7±2.1
1
2
HbO2
HbO2
HbO2
HbO2
HbO2
metHb
HbCO
-
1.5
1.5
1.5
1.5
1.5
1.5
1.5
-
(0.5)
1.0
(1.0)
1.5
(7.6±1.5)
12.6±1.0
(15.6±0.9)
14.8±1.8
(16.0±1.2)
16.3±0.8
(20.4±1.4)
1.3±0.4
3
(1.5)
3.0
4
(3.0)
15.0
5
(15.0)
1.5
6
(1.5)
1.5
(4.5±1.4)
0.5±0.3
(1.2±0.6)
1.0±0.6
7
(1.5)
1.5
(0.5±0.4)
(1.0±0.5)
3.4±0.6b
(3.6±0.4)b
3.9±0.7b
(4.1±0.8)b
20.2±0.9b
(19.2±0.6)b
20.6±0.6b
(19.9±0.8)b
N.D.d
1.1±0.7b
(1.1±0.7)b
1.2±0.7b
(1.3±1.6)b
1.0±0.6b
(0.6±0.6)b
1.7±0.7b
(1.6±0.5)b
8
(1.5)
3.0
9
-
-
(3.0)
30.0
10
11
-
-
(30.0)
45.0
-
-
(45.0)
-
12
13
HbO2
1.5
1.5
-
-
N.D.d
SFHL
-
20.1±1.4
21.6±1.7
(HbO2)c
a Mean±SD (n = 3). Solutions were diluted 1000 times for pseudo-enzymatic activity measurements.
b Assuming Hb concentration of 1.5 mM. c SFHL, stroma free hemolysate. d Not detected.
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Thermal Stability of NADH and NADPH—After aqueous solutions of NADPH and
NADH dissolved in phosphate buffered saline (PBS) were sealed in vials, they were incubated at
25, 30, 37, and 50 °C. The concentration of the reduced form NAD(P)H was measured
periodically by absorption at 340 nm. The initial absorbance of the NAD(P)H solutions was
defined as 100% recovery. The rate constant (k) of oxidation was calculated at each incubation
time. Based on those calculations, an Arrhenius plot was created (Figure 2). Activation energies
(Ea) were calculated respectively as 251 and 167 kJ/mol for NADH and NADPH. Table 2
presents the times necessary to reach 80 and 20% recoveries of NADH and NADPH. Apparently,
NADH is more thermally stable than NADPH. For example, NADH requires 116 days to be
oxidized to 20% recovery in a physiological aerobic condition, whereas NADPH requires only
1.2 days.
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Figure 2. Arrhenius plot of rate constants (k) for the oxidation of
NADH and NADPH in PBS pH 7.4 buffer.
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Table 2. Oxidation of NADH and NADPH in PBS (pH 7.4, 25 or 37 °C).
Symbols t0.8 and t0.2 respectively denote the times necessary to reach 80%
and 20% recoveries.
7
Potential
t0.8 at 25 °C
t0.8 at 37 °C
t0.2 at 37 °C
8
9
Antioxidant
(day)
(day)
(day)
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60
NADH
834
2.3
16
116
1.2
NADPH
0.2
Michaelis Constants of the Pseudo-enzymatic Activities and Influence of IHP and
DPG—Lineweaver–Burk plots (Figure 3) for NAD(P)H coexisting with HbO2 were created from
the data in Table 1. The Michaelis constants (Km) were calculated from intersection with the X-
axis. The Km values for pseudo-CAT are 1.4 mM (NADPH) and 1.8 mM (NADH). Furthermore,
those for pseudo-SOD are 2.1 mM (NADPH) and 2.3 mM (NADH). Equimolar addition of IHP
or DPG to NAD(P)H coexisting with HbO2 showed competitive inhibition patterns with
consistent intersections with the Y-axis while the slope increased, indicating that the Vmax values
were consistent and that the apparent Km increased. The inhibition dissociation constant (Ki) of
IHP was higher than that for DPG. Results show that IHP inhibited the pseudo-enzymatic
activities more strongly than DPG did (Table 3).
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Figure 3. Lineweaver–Burk plots of pseudo-CAT and pseudo-SOD activities for NAD(P)H (1–
15 mM) in the presence of 1.5 mM HbO2, with or without equimolar IHP or DPG as inhibitors.
VCAT, Pseudo-CAT, ×104 IU/g Hb; VSOD, Pseudo-SOD, ×102 U/g Hb.
Effects of NAD(P)+ and PLP on Pseudo-CAT and SOD Activities—Inhibitory effects
of oxidized forms NAD+ and NADP+, and PLP on the pseudo-enzymatic activities of NAD(P)H
coexisting with HbO2 were examined by adding them at the equimolar concentration of
NAD(P)H to create Lineweaver–Burk plots (data not shown). As a result, both Vmax and Km were
unaffected by the addition of NAD+, NADP+, and PLP, as presented in Table 3.
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Table 3. Kinetic parameters of the pseudo-CAT and pseudo-SOD activities of NAD(P)H with
coexistence with HbO2, and the influence of the potential inhibitors: IHP, DPG, NAD+, NADP+,
and PLP.
Potential
Pseudo-
Potential
Km
Vmax
Ki
7
8
9
Antioxidant enzymatic Inhibitor
(mM)
(CAT, ×104IU/g Hb)
(SOD, ×102U/g Hb)
(mM)
activity
None
IHP
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23
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25
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1.8
8.8 a
3.2 a
2.0 a
2.2 a
2.3
11.0 a
4.1 a
2.2 a
2.1 a
1.4
7.8 a
2.9 a
1.4 a
1.4 a
2.1
6.8 a
4.7 a
2.0 a
1.8 a
56.2
56.5
55.6
56.8
56.5
27.4
31.8
26.3
28.2
29.0
52.9
52.4
52.6
54.6
54.3
22.4
18.6
21.6
22.1
21.6
-
2.1
9.6
-
CAT
SOD
CAT
SOD
DPG
NAD+
PLP
-
NADH
None
IHP
-
1.7
8.6
-
DPG
NAD+
PLP
-
None
IHP
-
2.4
9.9
-
DPG
NADP+
PLP
-
NADPH
None
IHP
-
3.2
5.8
-
DPG
NADP+
PLP
-
a Apparent Michaelis constant in the presence of a potential inhibitor.
HPLC Analyses of the Autoxidation Intermediate Hybrids of HbO2 in the Presence of
NADH—During autoxidation of the HbO2 solution (1.5 mM) in PBS in the absence of NADH,
+
the chromatogram showed increases of partially oxidized intermediate hybrid signals, α2 β2
+
+
+
(elution time: 2.0 min) and α2β2 (2.3 min), and then the totally oxidized α2 β2 (2.5 min) (Figure
+
+
4A). The ratio of the peak area of α2 β2 to that of α2β2 was as high as 1.2 throughout the
measurement, indicating that α-subunit heme is more prone to be autoxidized (Figure 4B). On
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the other hand, in the presence of 1.5 mM NADH, the total levels of the partially and totally
5
6
7
oxidized Hbs were considerably suppressed as compared with those without NADH (Figure 4A),
8
9
+
+
and the ratio of the peak area of α2 β2 versus that of α2β2 was lower than 0.9, indicating that
NADH preferentially hindered autoxidation of α-subunit heme, presumably because NADH is
located closer to α-subunit heme than to β-subunit heme.
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Figure 4. (A) HPLC profiles of the intermediate Hb hybrids during autoxidation of HbO2 at 37
oC in the presence (+) and absence (-) of NADH (1.5 mM) in PBS. (B) Time courses of the peak
+
+
area ratio of α2 β2 versus that of α2β2 , calculated from the chromatograms in (A).
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31P NMR of NADH in the Presence of HbO2—NADH possesses two phosphate groups
between nicotinamide and adenine but shows a singlet strong peak at around -11 ppm at the
magnetic field employed, which is far different from the peak of phosphate (2.2 ppm) derived
from PBS (data not shown). By the addition of HbO2, the peak intensity decreased while other
three upfield peaks appeared (Figure 5A). Because of the presence of irons in Hb, all the peaks
tended to be left-shifted in parallel to a lower field. In the presence of equimolar amount of 1.5
mM HbO2, the spectrum of NADH is composed of the AB quartet split pattern with JAB = 20 Hz
and AB = 0.3 ppm, indicating that the two phosphate groups in the NADH molecule are located
in different environments, presumably induced by the binding with HbO2. From the integrated
intensities of the signals, the percentage of the amount of NADH bound with HbO2 was
calculated for all the analytes (Figure 5B), considering the overlapping of the singlet signal of
free NADH and the split leftmost signal of interacted NADH. The amount of NADH bound with
HbO2 proportionally increased with the amount of HbO2. The results indicate the 1:1
stoichiometric binding of NADH to HbO2.
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Figure 5. (A) The proton-decoupled 31P NMR of NADH (1.5 mM) in the presence of HbO2 (0.5,
1.0, and 1.5 mM) in PBS. (B) The percentage of NADH bound with HbO2 with increaseing the
amount of HbO2.
Docking Simulation of NAD(P)H and HbO2—Atomic coordinates of HbO2 and
NAD(P)H were obtained from Protein data bank (PDB) registered as X-ray structural analysis. A
docking simulation was conducted using MF myPresto v3.2 mmMPApp2 application software
(Figure 6). The interatomic distances of major sites are portrayed in Table 4. Reportedly, DPG
binds to HbO2 with interatomic distance of 2.57–2.89 Å between phosphate groups of DPG and
α-subunit Lys99 and Arg141.12 Our simulation clarified that both NADH and NADPH bind to the
same position on HbO2 with interatomic distance of 1.85–4.08 Å between NADH and α-subunit
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Lys99 and Arg141 (Figure 6), and 2.42–3.63 Å between NADPH and the same residues (see
Supporting Information). In Table 4, the set of oxygens (O8, O9) in NADH corresponds to one
phosphate group near nicotinamide locating nearby α2Lys99, and another phosphate group near
nicotinamide is apparently distant from the residue. Such different environments of two
phosphate groups should affect their 31P NMR. The interatomic distance between the carbon (C5)
in a heme and the carbon (C19) of nicotinamide is 10.08 Å for NADH (Figure 6) and 9.24 Å for
NADPH (see Supporting Information). We also simulated docking NADH to deoxyHb, which
revealed that NADH binds to β-subunits, not to α-subunits, and that the distances from NADH to
the β-subunit Lys82 were 2.95 Å, and to the β-subunit His143 was 3.44 Å, although the
interatomic distance to the carbon (C19) of the heme structure was estimated as 19.99 Å for β1-
heme (see Supporting Information).
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Figure 6. Proposed interacting sites between HbO2 and NADH
obtained from the docking simulation using a MF myPresto system.
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Table 4. Interatomic distances (Å) between the ligand sites and
protein sites (HbO2) calculated from the docking simulation using a
MF myPresto system
Distance (Å)
Ligand
Ligand-site (A)
HbO2-site (B)
between (A)–(B)
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NADH-O8
NADH-O9
NADH-O11
NADH-O12
NADH-O14
NADH-O14
NADH-C19
NADPH-O1
NADPH-O8
NADPH-O9
NADPH-O10
NADPH-O12
NADPH-O13
NADPH-C19
DPG-O2
α2Lys99-NZ
α2Lys99-NZ
α1Lys99-NZ
α1Lys99-NZ
α2Arg141-NH2
α2Arg141-NZ
α2HEM-C5
3.49
2.10
3.40
4.08
1.85
2.58
10.08
2.61
3.63
2.42
2.83
2.82
3.48
9.24
2.89
2.87
2.86
2.66
2.57
2.64
NADH
α1Lys99-NZ
α2Lys99-NZ
α1Lys99-NZ
α2Lys99-NZ
α2Arg141-NZ
α2Arg141-NZ
α2HEM-C5
NADPH
α1Lys99-NZ
α2Arg141-NH2
α2Lys99-NZ
α2Lys99-NZ
α2Arg141-NH1
α2Arg141-NH2
DPG-O5
DPG-O10
DPGa
DPG-O11
DPG-O13
(Wat639-O)
a Referred from results of an earlier study.12
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DISCUSSION
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NADH within RBCs contributes to reduction of metHb as the coenzyme of NADH-
cytochrome b5 reductase. However, our earlier report described that a high concentration of
NADH coexisting with purified HbO2 showed a reduced rate of autoxidation at 37 °C for 24 h in
the absence of the corresponding enzymes.8 An analogic molecule NADPH showed a markedly
weaker effect even though both NADH and NADPH possess equal standard redox potential (E’0
= – 0.32 V). The reason for this different effect of NADPH versus NADH was not clarified in our
previous study. In the present study, we demonstrated that rapid metHb formation induced by the
oxidants (H2O2, NO, NaNO2) was suppressed by the addition of NADPH, with effects similar to
those of NADH. Furthermore, the pseudo-CAT and pseudo-SOD activities of NADPH in the
presence of HbO2 were nearly equal to those of NADH. Nevertheless, a difference was found
between NADH and NADPH in terms of thermal stability. For example, in an aerobic condition
at 37 °C NADH requires 116 days for oxidization to 20% recovery, although NADPH requires
only 1.2 days. The respective activation energies (Ea) of oxidation for NADPH and NADH are 40
and 60 kcal/mol, indicating that NADPH is very unstable. We speculate that this thermal
instability is the reason that NADPH showed insufficient suppression of metHb formation over a
long period of time (24 h), although both have similar pseudo-enzymatic activities in the presence
of HbO2 that are readily measurable (< 1 h).
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The standard redox potential (E’0) of both NADH and NADPH is –0.32 V. However, in
some physiological conditions they show different redox potentials (E’).27,28 E’ of NADH is –
0.241, –0.280, or –0.318 V and that of NADPH ranges lower values; –0.370, –0.393, or –0.415
V. This difference indicates that NADPH is more prone to be oxidized. Reportedly, in an aerobic
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condition NAD(P)H initially reacts with oxygen to form NAD(P)H-peroxide anion, –O2-
NAD(P)H, which then capture a hydrogen ion to form hydrogen peroxide and oxidized
NAD(P)+.26 The generated hydrogen peroxide then reacts with another NAD(P)H to form
NAD(P)+ and water molecule. Wu et al.29 suggested that the different reactivity with hydrogen
ion between –O2-NADH and –O2-NADPH should cause the different stability. Because ionic
strength significantly affects the half-life of NADH, it is suggested that additional phosphate
group present on adenosine in NADPH should influence on the local ionic strength and stabilize
the oxidized pyridinyl cation of nicothinamide, that may shorten the half-life of NADPH.
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Next, we analyzed the kinetic parameters of the pseudo-enzymatic activities such as the
Michaelis constant (Km). The Km values were clarified to be of millimolar order. In the HbO2
solution (10 g/dL, 1.55 mM), the amount of NAD(P)H necessary for the inhibition of metHb
formation must be at least equimolar to HbO2 (Table 1). In contrast, the Km value of cytochrome
b5 reductase (metHb reductase system) with NADH as a coenzyme is reportedly 1.6 µM,30
demonstrating that Km of the NADH-HbO2 system is about 1,000 times higher. However, co-
encapsulation of such a high concentration of NADH is possible to prepare artificial red cells
(liposome-encapsulated Hbs) generating the pseudo-enzymatic activities sufficient to inhibit
metHb formation.8
The presence of two inhibitors (IHP, DPG) in the NAD(P)H-HbO2 pseudo-enzymatic
system indicated competitive inhibition because Vmax did not change although Km increased. X-
ray structure analysis clarified that both IHP and DPG bind to the cleft of β-subunits in the tense
state deoxyHb.31,32 However, a docking simulation report described they can also interact with
relaxed state HbO2.12,33 Therefore, it was inferred that interaction sites on HbO2 for NAD(P)H are
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the same as those for IHP and DPG.
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We tried to quantify the interaction of NAD(P)H and HbO2 using isothermal titration
calorimetry, observing the generated heat by mixing two aqueous solutions. However, the heat
was so low as to subtract the dilution heat for sufficient validation (data not shown). This implies
that the interaction is essentially weak and their binding constants (Kb) are small. Actually,
Cashon et al.34 measured Kb of NADPH to HbO2 by a subtle shift of oxygen affinity, and it was
as small as 0.073 mM-1. However, they failed to measure Kb of NADH, though they obtained
much higher Kb for NAD(P)H to deoxyHb. Ogo et al.35,36 measured Kb by fluorescence titrations
of NAD(P)H and obtained Kb for deoxyHb, but not for HbO2 because of the weak interaction. On
the other hand, our data of Lineweaver-Burk plots in the presence of an equimolar inhibitor (IHP
or DPG) to NAD(P)H (Figure 3) indicate that the inhibitor and NAD(P)H compete with each
other to bind to the same site on HbO2. Therefore, the Kb values for NAD(P)H would be
presumably comparable to or lower than those of IHP (2–10 mM-1) and DPG (0.4–1.2 mM-1)
reported in the literature.11,12 Addition of oxidized form NAD+ or NADP+ to NAD(P)H
coexisting HbO2 showed no inhibition effect to the pseudo-enzymatic activities, indicating that
neither NAD+ nor NADP+ binds to the NAD(P)H binding site. Accordingly, after the NAD(P)H-
HbO2 system shows pseudo-enzymatic activities, the oxidized NAD(P)+ dissociates immediately.
It is replaced with new NAD(P)H for repeated pseudo-enzymatic activity. Actually, PLP is used
as an allosteric effector for Hb in liposome-encapsulated Hbs (artificial red blood cells).8 It is
known to bind to the terminals of α-chains and β-chains,37 which is different from the binding site
of NAD(P)H, explaining why PLP showed no inhibition effect.
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31P NMR is widely used for the analysis of weak interaction of phosphate compounds
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with proteins.38,39 31P NMR of NADH provided a direct evidence that NADH stoichiometrically
interacts with HbO2 at 1:1 molar ratio as shown in Figure 5. Even though NADH possesses two
phosphate groups, the unbound free NADH shows a singlet signal. On the other hand, the
interacted NADH showed quartet split signals, indicating that the environments of the two
phosphate groups are different after binding to HbO2. Reportedly the reason for the fact that
oxidized NAD+ shows similar quartet split signals is that the oxidized nicotinamide contains a
charged nitrogen in the pyridine ring and that there is an intramolecular electrostatic interaction
between this nitrogen and the negatively charged phosphate.40 Therefore, in our study it is
speculated that the phosphate groups in NADH electrostatically interacts with positive charges on
the globin chain of HbO2, inducing heterogeneous environments for the two phosphate groups.
The reasons why NAD+ does not interact with HbO2 are that the positive charge of NAD+ hinders
the binding to the positively charged site of globin or that the molecular conformation of NAD+ is
distorted because of the intramolecular electrostatic interaction as mentioned above.
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The binding site of IHP and DPG on HbO2 is located near the α-subunit heme.12,33
Presumably, NAD(P)H should preferentially interacts at the same site, which might be related to
the antioxidative property of NAD(P)H coexisting with HbO2 and the consequent inhibition of
metHb formation. The acceleration of disproportional reaction (Eq. 1) by the pseudo-SOD
activity and the elimination of (O2·–) are expected to contribute to the reduced metHb formation
reaction (Eq. 2) by H2O2.8
2O2·– + 2H+ → H2O2 + O2
(Eq. 1)
(Eq. 2)
2Hb(Fe2+)O2 + H2O2 + 2H+ → 2metHb(Fe3+) + 2H2O + O2
Regarding the mechanism of pseudo-CAT activity, the following reaction system can be
considered (Eqs. 3 and 4). First, metHb reacts to H2O2, generating ferryl Hb radical (Fe4+=O),
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which in turn interacts with NADH, providing it with two electrons. Then H2O2 is eliminated
while metHb is reproduced.41
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H-metHb(Fe3+) + H2O2 → ∙ferrylHb(Fe4+=O) + H2O + H+
(Eq. 3)
(Eq. 4)
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∙ferrylHb(Fe4+=O) + NADH + 2H+ → H-metHb(Fe3+) + NAD+ + H2O
We have been trying crystallization of HbO2 in the presence of NADH for the detailed
structural analysis by X-ray crystallography. However, it is not successful so far probably
because of the very weak interaction between NADH and HbO2. Laberge et al.,12 conducted
structural analysis using docking simulation on phosphate compounds such as 2,3-DPG and IHP
which are weakly bound to the relax state HbO2. Therefore, we have no choice but to test docking
simulation. Our docking simulation of NADH and HbO2 estimated the binding sites of NADH on
HbO2. The interatomic distance of DPG (phosphate group) and HbO2 (α-subunit Lys99, Arg141),
obtained with docking simulation by Laberge et al., is reportedly 2.57–2.89 Å.12 As shown in
Figure 6, the interatomic distance of NADH and the amino acid residues of HbO2 was 1.85–4.08
Å, which surely allows potential binding. The interatomic distance between the carbon (C5) in α-
subunit heme skeleton and the carbon (C19) of nicotinamide site relating to the redox was 10.08
Å. This is a long distance, but it has been presented in some reports of the literature42-44 that
electron transfer is possible even at 10.5 Å in perturbing proteins, supporting the potential
electron transfer from NADH to a heme over this interatomic distance. In addition, the
interatomic distance between NADPH and the amino acid residues of HbO2 was estimated as
2.42–3.63 Å. That of a heme and nicotinamide is 9.24 Å. These interatomic distances of NADPH
are equivalent to those of NADH. When oxidizing substances such as ROS approach a heme
iron, the oxidation of heme can be inhibited by the reducing power of adjacent NAD(P)H bound
to HbO2. Actually, ROS is expected to be eliminated by pseudo-SOD and pseudo-CAT; metHb
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formation was inhibited by this effect.
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Because the binding site of NAD(P)H on HbO2 seems preferentially in α-subunits and
not in β-subunits, it is expected that the oxidation of β-subunit heme would be less inhibited.
Actually, HPLC profiles of the metHb intermediates during autoxidation showed relatively
higher degree of inhibitory effect of NADH on α-subunit heme as shown in Figure 4, supporting
that NADH locates near the α-subunit heme.
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The docking simulation of deoxyHb and NADH clarified that the binding residues are β-
subunit Lys82 and His143 with interatomic distances of 2.95 and 3.44 Å, respectively (see
Supporting Information). In addition, the Kb value for deoxyHb is presumably higher than that
for HbO2.34-36 However, the interatomic distance between heme and NADH is as long as 20 Å;
this distance would allow electron transfer between them only to a slight degree. The
measurement of pseudo-enzymatic activities cannot be done in a deoxygenated condition because
of the generation of dioxygen in the analyte solution. However, it is speculated that the pseudo-
enzymatic activities of NAD(P)H coexisting with deoxyHb are low in comparison to those of
HbO2.
Intraerythrocytic concentrations of NAD+ and NADH are 6.5×10-2 mM and 2.8×10-4
mM, respectively.9 Those of NADP+ and NADH are 6.5×10-5 mM and 6.5×10-2 mM,
respectively. The amount of NAD(P)H bound to Hb (5.4 mM) should be very small. The
physiological role of the pseudo-enzymatic activities of the combination of NAD(P)H-HbO2 in a
normal healthy condition is not known, however, it would be conceivable that such antioxidative
function works in a genetic disease such as deficiency of ROS-eliminating enzymatic systems.
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