Pyrroloquinoline quinone (PQQ) is reduced to pyrroloquinoline quinol (PQQH₂) by vitamin C, and PQQH₂ produced is recycled to PQQ by air oxidation in buffer solution at pH 7.4

Abstract

Measurements of the reaction of sodium salt of pyrroloquinoline quinone (PQQNa₂) with vitamin C (Vit C) were performed in phosphate-buffered solution (pH 7.4) at 25 °C under nitrogen atmosphere, using UV–vis spectrophotometry. The absorption spectrum of PQQNa₂ decreased in intensity due to the reaction with Vit C and was changed to that of pyrroloquinoline quinol (PQQH₂, a reduced form of PQQ). One molecule of PQQ was reduced by two molecules of Vit C producing a molecule of PQQH₂ in the buffer solution. PQQH₂, thus produced, was recycled to PQQ due to air oxidation. PQQ and Vit C coexist in many biological systems, such as vegetables, fruits, as well as in human tissues. The results obtained suggest that PQQ is reduced by Vit C and functions as an antioxidant in biological systems, because it has been reported that PQQH₂ shows very high free-radical scavenging and singlet-oxygen quenching activities in buffer solutions.

Graphical abstract

Recycling of PQQH₂ and PQQ under the coexistence of Vit C.

Key words:

• pyrroloquinoline quinone
• pyrroloquinoline quinol
• reaction rate
• UV–vis absorption spectrum
• vitamin C

Pyrroloquinoline quinone (PQQ) has attracted much attention in the recent years owing to its several interesting physiological functions.^1,2) PQQ is a water-soluble quinone compound first identified as a cofactor of alcohol and glucose dehydrogenases in bacteria.^3,4) PQQ also functions as a growth factor and contributes to mammalian cell growth.^5–7) It has been also reported to show neuroprotective effects.^8–10) A recent study suggests that PQQ protects the spinal cord against secondary damage by attenuating the inducible nitric oxide synthase expression following a primary physiological injury.¹¹⁾

The reduced form of PQQ, i.e., PQQH₂ (pyrroloquinoline quinol, see Fig. 1) exhibits antioxidative properties in in vitro examinations.^12,13) In experiments using cultured cells, it has been reported that PQQ prevents oxidative stress-induced neuronal death.^10,14) Moreover, a marked decrease in ischemia damage was observed for in vivo models such as in the case of cardiovascular or cerebral ischemia.^9,15) PQQ preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes.¹⁶⁾ Furthermore, it has been reported that PQQ prevents cognitive deficit caused due to oxidative stress in rats.^17,18)

Fig. 1. Molecular structures of PQQNa₂, PQQH₂, vitamin C (ascorbic acid, AsH₂), and ascorbate monoanion (AsH⁻).

Small amounts of PQQ have been found in many kinds of foods such as fruits and vegetables.^2,19–21) Furthermore, the presence of small amounts of free PQQ has also been found in eight of the human organs, in plasma and urine and in three of the rat organs.^22,23)

Earlier PQQH₂ has been prepared by the reaction of PQQ with thiols (RSH) (such as thiophenol, mercaptoethanol, cysteine, and 1,4-butanedithiol)) in 0.05 M acetate buffer (including 20% CH₃CN) under anaerobic conditions (see reaction (1(1) $$\text{PQQ}+2\text{RSH}\to {\text{PQQH}}_2+\text{RSSR}$$(1) )).²⁴⁾ Reduction of PQQ to PQQH₂ has also been performed using dithiothreitol with two SH groups in the same molecule.²⁵⁾ Furthermore, we found that PQQNa₂ (disodium salt of PQQ) could be easily reduced to PQQH₂ by reacting it with cysteine and glutathione (RSH) in 0.05 M phosphate-buffered solution (pH 7.4) under nitrogen atmosphere (Fig. 1).²⁶⁾ These results suggest that PQQ exists as a reduced form within the cells and plays an important role as an antioxidant (AO).(1) $$\text{PQQ}+2\text{RSH}\to {\text{PQQH}}_2+\text{RSSR}$$(1)

It is well known that lipid peroxyl radical (${\text{LOO}}^{\cdot }$) and singlet oxygen (¹O₂) are two major representative reactive oxygen species generated in biological systems. In a previous work, aroxyl radical (${\text{ArO}}^{\cdot }$)-scavenging activities of PQQH₂ and water-soluble AOs (such as vitamin C (Vit C) and uric acid) were measured in 5.0 wt% Triton X-100 micellar solution (pH 7.4) using stopped-flow spectrophotometry (see reaction (2$$k_s$$)).²⁶⁾ A stable aroxyl radical (${\text{ArO}}^{\cdot }$) (2,6-di-t-butyl-4-(4′-methoxyphenyl)phenoxyl) was used as a model for ${\text{LOO}}^{\cdot }$ radical.^27,28) The second-order rate constant (k_s) (1.86 × 10³ M⁻¹ s⁻¹) for the reaction of ${\text{ArO}}^{\cdot }$ with PQQH₂ was found to be 7.4 times larger than that of Vit C (2.51 × 10² M⁻¹ s⁻¹), which is known as the most active water-soluble AO.$$k_s$$(2) $${\text{ArO}}^{\cdot }+{\text{PQQH}}_2\to \text{ArOH}+{\text{PQQH}}^{\cdot }$$(2)

Furthermore, a kinetic study of the quenching reaction of ¹O₂ with PQQH₂ and other seven natural AOs (Vit C, uric acid, epicatechin, epigallocatechin, α-tocopherol (α-TocH), ubiquinol-10 (UQ₁₀H₂), and β-carotene) (reaction (3(3) $${}^1{\text{O}}_2+{\text{PQQH}}_2\to {}^3{\text{O}}_2+{\text{PQQH}}_2$$(3) )) has been performed in 5.0 wt% Triton X-100 micellar solution (pH 7.4), indicating that PQQH₂ shows high ¹O₂-quenching activity.²⁹⁾$$k_Q$$(3) $${}^1{\text{O}}_2+{\text{PQQH}}_2\to {}^3{\text{O}}_2+{\text{PQQH}}_2$$(3)

These results suggest that PQQH₂ may contribute to the protection of oxidative damage in biological systems by scavenging the free-radicals and quenching ¹O₂.

As described above, Vit C is well known as a representative water-soluble AO. Vit C (ascorbic acid (AsH₂)) will take the ascorbate monoanion (AsH⁻) structure at pH 7.4 (see Fig. 1).³⁰⁾ Hydrophilic Vit C (AsH⁻) enhances the AO activity of α-TocH by regenerating α-tocopheroxyl ($\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }$) radical to α-TocH (reaction (4$$k_Q$$))(4) $$\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }+{\text{AsH}}^{-}\to \mathit{\alpha }\text{-}\text{TocH}+{\text{As}}^{-\cdot }$$(4)

where ${\text{As}}^{-\cdot }$ is ascorbate free-radical. The reaction of $\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }$ with Vit C is well known as the usual tocopherol-regeneration reaction in biomembrane systems.

PQQ and Vit C are found in a number of foods^{2,19–21,31)} in addition to human and rat tissues.^{22,23,32–34)} High concentrations of Vit C are included in human plasma.³²⁾ In this study, the UV–vis absorption measurements for the reaction of PQQNa₂ (that is, PQQ) with Vit C were performed in 0.05 M phosphate-buffered solution (pH 7.4) at 25.0 °C. Absorption spectrum of PQQNa₂ was decreased by the reaction with Vit C and changed to that of PQQH₂ (see reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) )). Our results suggest that PQQ is reduced by Vit C lacking the SH group and functions as an AO in biological systems.(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5)

Materials and methods

Materials

Commercial Vit C (Wako Chemicals, Japan) was used as received. Powdered sample of PQQNa₂ was supplied by Mitsubishi Gas Chemical Co., Inc., Japan. The results for the elemental analysis, thermogravimetry, and titration of H₂O using the Karl-Fischer’s reagent indicated that PQQNa₂ was present in the monohydrate form (${\text{PQQNa}}_2^{\cdot }{\text{H}}_2\text{O}$).²⁶ The buffer solution was prepared using the distilled water treated with a Millipore Q system, and the pH was adjusted to 7.4 using 0.05 M KH₂PO₄–Na₂HPO₄ solution.

Methods

Measurements of the UV–vis absorption spectrum for the reaction of PQQNa₂ with Vit C (reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) )) were performed using a Shimadzu UV-2100S spectrophotometer by mixing equal volumes of 0.05 M phosphate-buffered solutions (pH 7.4) of PQQNa₂ and Vit C. Generally, 0.01 M phosphate-buffered (pH 7.4) solution is used for the biochemical experiments. Vit C is a weak acid, as it is well known. However, in the present work, comparatively high concentrations of Vit C (5.36 × 10⁻⁴ and 5.36 × 10⁻³ M) were used for the measurements. Consequently, 0.05 M phosphate-buffered (pH 7.4) solution was used for the measurements to avoid the shift of pH value.

PQQNa₂ is stable, and Vit C is comparatively stable in the buffer solution (pH 7.4) under air. On the other hand, as reported in previous works, PQQH₂ is unstable in the buffer solution (pH 7.4) under air and easily oxidized to PQQ.^26,35,36) Consequently, the reduction of PQQNa₂ to PQQH₂ by Vit C was performed under strictly de-aerated and nitrogen-substituted conditions using a Hamilton 1000 series gas-tight syringe and sealing cap to avoid oxidation of PQQH₂. Air oxidation of PQQH₂ was performed by taking off the above sealing cap from quartz cell, and then, quartz cell containing the reaction mixture was covered with sponge rubber cap to avoid the evaporation of buffer solution. The details of the UV–vis absorption measurements are the same as reported in a previous study.²⁶⁾ All the measurements were performed at 25.0 ± 0.5 °C.

Results

UV–vis absorption spectra of PQQNa₂, PQQH₂, and Vit C in buffer solution at pH 7.4

UV–vis absorption spectra of PQQNa₂ and Vit C were measured in 0.05 M phosphate-buffered solution at pH 7.4, as shown in Fig. 2. The maximum value of the absorption peaks (λ_max) and values of molar extinction coefficient (ε_max) obtained for PQQNa₂ and Vit C are listed in Table 1 (where “sh” stands for shoulder).

Fig. 2. The UV–vis absorption spectra of PQQNa₂ (–––), PQQH₂ (·····), and Vit C (AsH⁻) (- - -) with the same concentrations of 4.45 × 10⁻⁵ M in 0.05 M phosphate-buffered solution (pH 7.4) at 25.0°.

Note: The spectra of PQQNa₂ and PQQH₂ were reported in a previous study (see Fig. 2 in Ref. [26]).

Table 1. Values of UV–vis absorption maxima (${\mathit{\lambda }}_{max}^i$) and molar extinction coefficients (${\mathit{\epsilon }}_{max}^i$) for PQQNa₂, PQQH₂, and Vit C in 0.05 M phosphate-buffered solution (pH 7.4).

	${\mathit{\lambda }}_{max}^1$/nm (ε^1/M^−1 cm^−1)	${\mathit{\lambda }}_{max}^2$/nm (ε^2/M^−1 cm^−1)	${\mathit{\lambda }}_{max}^3$/nm (ε^3/M^−1 cm^−1)	${\mathit{\lambda }}_{max}^4$/nm (ε^4/M^−1 cm^−1)	${\mathit{\lambda }}_{max}^5$/nm (ε^5/M^−1 cm^−1)	${\mathit{\lambda }}_{max}^6$/nm (ε^6/M^−1 cm^−1)
PQQNa2	307.5 sh^a^b (10,800)	304.0 sh^b (11,100)	249^c (26,600)	267 sh^c (20,500)	331^c (12,700)	477^c (692)
PQQH2	307.5 sh^b (37,700)	304.0^c (40,000)	340 sh^c (11,500)	405 sh^c (2410)	499^c (1170)
Vit C	307.5 sh (82.6)	304.0 sh (193)	266.0 (15,200)

^a sh: shoulder.

^b The λ_max and ε_max values of PQQNa₂ and PQQH₂ at 307.5 and 304.0 nm were estimated from the UV–vis absorption spectra shown in Fig. 2.

^c The λ_max and ε_max values of PQQNa₂ and PQQH₂ in 0.05 M phosphate-buffered solution (pH 7.0) were reported in a previous study (Ref. [26]).

In a previous work, the reduction of PQQNa₂ to PQQH₂ was performed using cysteine and glutathione in 0.05 M phosphate-buffered solution at pH 7.4 (reaction (1(1) $$\text{PQQ}+2\text{RSH}\to {\text{PQQH}}_2+\text{RSSR}$$(1) )).²⁶⁾ The UV–vis absorption spectra of PQQH₂ obtained by the above reductions were similar to each other. The absorption spectrum of PQQH₂ obtained by the reduction using cysteine is shown in Fig. 2, together with those of PQQNa₂ and Vit C. The spectra of PQQH₂, PQQNa₂, and Vit C with the same concentration of 4.45 × 10⁻⁵ M in buffer solution are shown in Fig. 2. The values of λ_max and ε_max reported for PQQH₂ are listed in Table 1.

The values of the molar extinction coefficients (ε_304.0 and ε_307.5) at λ = 304.0 and 307.5 nm for PQQH₂, PQQNa₂, and Vit C are also listed in Table 1, as these values are necessary to analyze the changes in the UV–vis absorption spectra observed by the reaction between PQQNa₂ and Vit C (reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) )).

PQQNa₂ is reduced to PQQH₂ by the reaction with Vit C in buffer solution at pH 7.4

The measurements for the reaction of PQQNa₂ with Vit C were performed in a buffer solution. Fig. 3, panels (A) and (B), shows an example of the reaction between PQQNa₂ (4.45 × 10⁻⁵ M) and Vit C (5.36 × 10⁻⁴ M) in 0.05 M phosphate-buffered solution (pH 7.4) at 25.0 °C. The spectra were recorded at (A) 6 min (t = 0–90 min) and (B) 3 h (t = 1.5–15.5 h) intervals. As shown in the Fig. 3(A) and (B), notable changes in the absorption spectrum of PQQNa₂ were observed indicating the reaction of PQQNa₂ with Vit C. Two isosbestic points were clearly observed at 342 and 396 nm, suggesting that the reaction is simple and the contribution of any side reactions is negligible.

Fig. 3. Changes in the absorption spectrum of PQQNa₂ and PQQH₂ during the reaction of PQQNa₂ with Vit C in 0.05 M phosphate-buffered solution (pH 7.4) at 25.0 °C.

Notes: [PQQNa₂]_t = 0 = 4.45 × 10⁻⁵ M. [Vit C]_t = 0 = 5.36 × 10⁻⁴ M. The spectra were recorded at (A) 6-min interval and (B) 3-h interval. The downward arrow indicates a decrease (↓) (PQQNa₂), and an upward arrow indicates an increase (↑) (PQQH₂) in the absorbance with time.

As reported in a previous study, a decrease (↓) in the absorbance of PQQNa₂ at ~370 nm and an increase in the absorbance of PQQH₂ at 304 nm (see Fig. 4 in Ref. [26]) was observed in the reactions of PQQNa₂ with cysteine and glutathione in buffer solution (pH 7.4). This was because these reducing agents do not show any absorption at ~304 nm. Two isosbestic points (348 and 410 nm) were observed for the reaction of PQQNa₂ with glutathione. It was clear that PQQH₂ was produced as a result of the reaction of PQQNa₂ with cysteine and glutathione. Itoh et al. had reported similar results for the reaction of PQQ with thiophenol in acetate buffer (pH 4).^24,36)

Fig. 4. Formation of PQQH₂ due to the reaction of PQQNa₂ with Vit C, and decay of PQQH₂ in the presence of air.

Notes: (A) Time dependence of the absorbance of PQQH₂ at 307.5 nm, after the reduction of PQQNa₂ with Vit C in 0.05 M phosphate buffer (pH 7.4) at 25.0 °C under nitrogen atmosphere (○). [PQQNa₂]_t = 0 = 4.45 × 10⁻⁵ M and [Vit C]_t = 0 = 5.36 × 10⁻⁴ M. (B) PQQH₂ solution produced due to the reaction of PQQNa₂ with Vit C was exposed to air, after keeping the solution under nitrogen atmosphere for 4.5 day. A decrease in the absorbance of PQQH₂ at 307.5 nm was observed in the presence of air (○).

On the other hand, Vit C shows an absorption maximum at λ_max = 266 nm (ε_max = 15,200 M⁻¹ cm⁻¹) and a weak absorption (shoulder) at 304 nm (ε₃₀₄ = 193 M⁻¹ cm⁻¹) (see Table 1 and Fig. 2). When the same concentrations (4.45 × 10⁻⁵ M) of PQQNa₂ and Vit C were used for the reaction, the absorption of Vit C at 304 nm is observed to be weak or negligible in comparison with that of PQQH₂. This is because the ε₃₀₄ value of Vit C (193 M⁻¹ cm⁻¹) is more than two orders of magnitude smaller than the ε₃₀₄ of PQQH₂ (40,000 M⁻¹ cm⁻¹), as shown in Fig. 2. However, in the present work, the concentration of Vit C (5.36 × 10⁻⁴ M) that was used for the reaction was 12 times higher than that of PQQNa₂ (4.45 × 10⁻⁵ M). The result indicates that strong absorption of Vit C, which was shown by dotted line (- - - - -) in Fig. 3(A) and (B), overlaps to that of PQQH₂. Consequently, we could not observe an absorption peak of PQQH₂ at 304 nm.

On the other hand, the changes in the absorption spectra observed at wavelength >304 nm, such as, a decrease (↓) in the absorbance of PQQNa₂ at ~370 nm and an increase (↑) in the absorbance of PQQH₂ at 307.5 nm with time (Fig. 3(A) and (B)) are similar to that observed for the reaction of PQQNa₂ with glutathione. Furthermore, two isosbestic points (at 342 and 396 nm) were observed for the reaction of PQQNa₂ with Vit C, and these data are also similar to the one (348 and 410 nm) observed for the reaction of PQQNa₂ with glutathione in a previous work.²⁶⁾ These results suggest that PQQNa₂ was reduced to PQQH₂ by the reaction with Vit C. Detailed reason was explained in the Discussion section.

By reacting PQQNa₂ with Vit C, a most notable increase in the absorbance of PQQH₂ was observed at 307.5 nm, as shown in Fig. 3(A) and (B). Fig. 4(A) shows the time course of the increase in absorbance at 307.5 nm of PQQH₂. This increase was observed when a 0.05 M phosphate-buffered solution (pH 7.4) containing PQQNa₂ (2 × 4.45 × 10⁻⁵ M) was mixed with a 0.05 M buffer solution of Vit C (2 × 5.36 × 10⁻⁴ M) (1:1, v/v; final concentrations of PQQNa₂ (4.45 × 10⁻⁵ M) and Vit C (5.36 × 10⁻⁴ M)). As shown in Fig. 4(A), the absorbance of PQQH₂ at 307.5 nm increases rapidly from 0.544 at t = 0 min to ~1.1 at t = 30 min, and then increases gradually to 1.460 at t = 14 h. A detailed analysis of the change in the absorbance observed for PQQH₂ is discussed in the Discussion section.

The reaction between PQQNa₂ and Vit C was performed by keeping the concentration of PQQNa₂ ([PQQNa₂] = 4.45 × 10⁻⁵ M) constant and using a 10 times higher concentration of Vit C ([Vit C] = 5.36 × 10⁻³ M). Interestingly, the absorption spectrum of PQQNa₂ disappeared rapidly and changed to that of PQQH₂ (data not shown).

Time dependence of the absorbance (●) observed at 307.5 nm for PQQH₂ is shown in Fig. 5(A), together with that (○) observed for the reaction of PQQNa₂ with lower concentration of Vit C (Fig. 4(A)). As shown in Fig. 5(A), the absorbance at 307.5 nm increased rapidly from 1.378 at t = 0 min to ~2.10 at t = 10 min and further increased slowly to 2.302 at t = 15 h. The increase in absorbance from t = 0 to 10 min is much faster than that for the solution containing a lower concentration of Vit C (5.36 × 10⁻⁴ M). PQQ molecules are considered to be reduced easily to PQQH₂ in the presence of higher concentration of Vit C.

Fig. 5. Formation of PQQH₂ due to the reaction of PQQNa₂ with Vit C, and decay of PQQH₂ in the presence of air.

Notes: (A) Time dependence of the absorbance of PQQH₂ at 307.5 nm, after the reduction of PQQNa₂ with Vit C in 0.05 M phosphate buffer (pH 7.4) at 25.0 °C under nitrogen atmosphere. [PQQNa₂]_t = 0 = 4.45 × 10⁻⁵ M. [Vit C]_t = 0 = 5.36 × 10⁻⁴ M (○) and 5.36 × 10⁻³ M (●). (B) PQQH₂ solution produced by the reaction of PQQNa₂ with Vit C was exposed to air, after keeping the solution under nitrogen atmosphere for 4.5 day. A decrease in the absorbance of PQQH₂ at 307.5 nm was observed in the presence of air.

The reaction mixtures obtained after the reaction of PQQNa₂ ([PQQNa₂] = 4.45 × 10⁻⁵ M) with Vit C ([Vit C] = 5.36 × 10⁻⁴ M (or 5.36 × 10⁻³ M)) were kept for 4.5 days at 5 °C under refrigeration. During these 4.5 days, no change in the spectrum was observed indicating that PQQH₂ produced during the above reaction is stable under the nitrogen atmosphere.

Air oxidation of PQQH₂ under the coexistence of Vit C in buffer solution at pH 7.4

As described in one of the previous sections, PQQH₂ is stable in the presence of Vit C in buffer solution under nitrogen gas atmosphere. On the other hand, exposing the solution to air (i.e. molecular oxygen (³O₂)), remarkable changes in the absorption spectra were observed, as shown in Fig. 6. A downward arrow indicates a decrease (↓) in the absorbance of PQQH₂ at 307.5 nm and an upward arrow suggests an increase (↑) in the absorbance of PQQ at 370 nm with time. As shown in Fig. 6, two isosbestic points were observed at 342 and 396 nm. These wavelengths are the same as those (342 and 396 nm) observed for the reduction reaction of PQQ with Vit C (see Fig. 3)). The result indicates that the oxidation of PQQH₂ by ³O₂ is also a simple reaction, and the contribution of side reactions is negligible. Furthermore, the absorption of PQQ was clearly observed at t = 300 min, as shown in Fig. 6. It is clear that PQQ was regenerated by the reaction of PQQH₂ with ³O₂ (reaction (6(6) $${\text{PQQH}}_2+{}^3{\text{O}}_2(\text{Air})\to \text{PQQ}+{\text{H}}_2{\text{O}}_2$$(6) )).³⁶⁾ The nature of the changes in the absorption spectra observed around the wavelength region (~300–500 nm) is very similar to those shown in Fig. 3. The result clearly indicates that PQQH₂ is recycled to PQQ due to the reaction with ³O₂ in the buffer solution at pH 7.4 (see Fig. 7).^25,36)(6) $${\text{PQQH}}_2+{}^3{\text{O}}_2(\text{Air})\to \text{PQQ}+{\text{H}}_2{\text{O}}_2$$(6)

Fig. 6. PQQH₂ solution produced due to the reaction of PQQNa₂ ([PQQNa₂]_t = 0 = 4.45 × 10⁻⁵ M) with Vit C ([Vit C]_t = 0 = 5.36 × 10⁻⁴ M) was exposed to air, after keeping the solution under nitrogen atmosphere for 4.5 day.

Notes: The spectra were recorded at 36-min time intervals. A downward arrow indicates a decrease (↓) (PQQH₂), and an upward arrow indicates an increase (↑) (PQQNa₂) in the absorbance with time.

Fig. 7. Recycling of PQQH₂ and PQQ under the coexistence of Vit C.

By reacting PQQH₂ with air, the most notable decrease in the absorbance of PQQH₂ was observed at 307.5 nm, as shown in Fig. 6. Fig. 4(B) shows the time course of the decrease in the absorbance at 307.5 nm for PQQH₂ observed when the solution containing PQQH₂ was exposed to air. As shown in Fig. 4(B), the absorbance of PQQH₂ at 307.5 nm decreases gradually from 1.420 at t = 0 min to ~0.60 at t = 120 min and further decreases very slowly to 0.544 at t = 300 min.

Similarly, by exposing the reaction mixture containing PQQH₂ and a high concentration of Vit C ([Vit C] = 5.36 × 10⁻³ M) to air, the change in the absorption spectrum was observed. However, the observed change was not very remarkable (data not shown). Fig. 5(B) shows the time course of the decrease in the absorbance of PQQH₂ at 307.5 nm observed when the solution was exposed to air. The absorbance of PQQH₂ at 307.5 nm (indicated by ●) decreases slowly from 2.305 at t = 0 min to 1.465 at t = 38 h. The decrease in the absorbance of PQQH₂ was much slower in comparison with the reaction mixture containing lower concentration of Vit C ([Vit C] = 5.36 × 10⁻⁴ M) (indicated by ○). Not only the PQQ produced by the air oxidation of PQQH₂, but also the residual Vit C in the reaction mixture contribute to the absorbance observed at 307.5 nm. The changes in the absorbance of PQQH₂ are discussed in greater details in the Discussion section.

Discussion

UV–vis absorption spectra of ${\text{Vit C}}^{\cdot }$ (or ${\text{As}}^{-\cdot }$) and ${\text{PQQH}}^{\cdot }$ radicals in buffer solution at pH 7.4

In the previous studies, the acid dissociation constants (K_a) for the three COOH groups and one NH group in PQQ were determined by the measurement of the pH dependence of oxidation–reduction potentials (E_1/2).^37–39) The three COOH groups in PQQ molecule will take a monoanion form (COO⁻) at pH 7.4 (Fig. 1), because the pK_a1, pK_a2, and pK_a3 values of the three COOH groups and the pK_a4 value of the NH group in the PQQ molecule are reported to be 1.60, 2.20, 3.30, and 10.30, respectively. Consequently, both PQQNa₂ and PQQ should take a similar molecular structure in the buffer solution with pH 7.4 and exhibit similar UV–vis absorption spectra. Therefore, we have used an abbreviation, “PQQ,” instead of “PQQNa₂” as needed (see reactions (1(1) $$\text{PQQ}+2\text{RSH}\to {\text{PQQH}}_2+\text{RSSR}$$(1) ), (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ), etc.).

Vit C (ascorbic acid, AsH₂) is a dibasic acid and can exist in three different molecular forms, i.e., the undissociated form (AsH₂), monoanion form (AsH⁻), and dianion form (As²⁻), depending on the pH value (Fig. 1). Vit C will take a monoanion form (AsH⁻) at pH 7.4, as the pK_a1 and pK_a2 values of Vit C (AsH₂) are 4.17 and 11.57, respectively.^30,40) Hence, we have used an abbreviation, “AsH⁻,” instead of Vit C as needed (see reactions (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ), (7(7) $$\text{PQQ}+{\text{AsH}}^{-}\to {\text{PQQH}}^{\cdot }+{\text{As}}^{-\cdot }$$(7) ), and (8(8) $${\text{PQQH}}^{\cdot }+{\text{AsH}}^{-}\to {\text{PQQH}}_2+{\text{As}}^{-\cdot }$$(8) )).

As described in the Introduction, the reaction between PQQNa₂ and Vit C (AsH⁻) could simply be expressed as shown in reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ), which is very similar to the reaction between PQQNa₂ and RSH (reaction (1(1) $$\text{PQQ}+2\text{RSH}\to {\text{PQQH}}_2+\text{RSSR}$$(1) )).²⁶⁾ However, by reacting PQQNa₂ with AsH⁻, the following reactions (7(7) $$\text{PQQ}+{\text{AsH}}^{-}\to {\text{PQQH}}^{\cdot }+{\text{As}}^{-\cdot }$$(7) )–(10(10) $${\text{As}}^{-\cdot }+{\text{As}}^{-\cdot }+2{\text{H}}_2\text{O}\to {\text{AsH}}_2+\text{As}+2{\text{OH}}^{-}$$(10) ) will proceed competitively in the buffer solution (pH 7.4). First, reaction (7(7) $$\text{PQQ}+{\text{AsH}}^{-}\to {\text{PQQH}}^{\cdot }+{\text{As}}^{-\cdot }$$(7) ) will occur producing ${\text{PQQH}}^{\cdot }$ and ${\text{As}}^{-\cdot }$, followed by reaction (8(8) $${\text{PQQH}}^{\cdot }+{\text{AsH}}^{-}\to {\text{PQQH}}_2+{\text{As}}^{-\cdot }$$(8) ) producing PQQH₂ and ${\text{As}}^{-\cdot }$. Furthermore, ${\text{PQQH}}^{\cdot }$ radicals are considered to be consumed by the disproportionation reaction producing PQQ and PQQH₂ (reaction (9(9) $${\text{PQQH}}^{\cdot }+{\text{PQQH}}^{\cdot }\to \text{PQQ}+{\text{PQQH}}_2$$(9) )), as described below.(7) $$\text{PQQ}+{\text{AsH}}^{-}\to {\text{PQQH}}^{\cdot }+{\text{As}}^{-\cdot }$$(7) (8) $${\text{PQQH}}^{\cdot }+{\text{AsH}}^{-}\to {\text{PQQH}}_2+{\text{As}}^{-\cdot }$$(8) (9) $${\text{PQQH}}^{\cdot }+{\text{PQQH}}^{\cdot }\to \text{PQQ}+{\text{PQQH}}_2$$(9) (10) $${\text{As}}^{-\cdot }+{\text{As}}^{-\cdot }+2{\text{H}}_2\text{O}\to {\text{AsH}}_2+\text{As}+2{\text{OH}}^{-}$$(10)

If the ${\text{As}}^{-\cdot }$ radical is stable in the buffer solution (pH 7.4), the absorption of ${\text{As}}^{-\cdot }$ radical would overlap with those of PQQ, PQQH₂, and Vit C in the process of the reaction. Bielski et al.⁴¹⁾ had measured the UV–vis absorption spectrum of ${\text{As}}^{-\cdot }$ radical using the pulse radiolysis technique. In this study, the ${\text{As}}^{-\cdot }$ radical showed an absorption peak at λ_max = 360 nm (ε_max = 5000 M⁻¹ cm⁻¹) at pH 7.4. Furthermore, they reported that ${\text{As}}^{-\cdot }$ radicals decay rapidly, following reaction (10(10) $${\text{As}}^{-\cdot }+{\text{As}}^{-\cdot }+2{\text{H}}_2\text{O}\to {\text{AsH}}_2+\text{As}+2{\text{OH}}^{-}$$(10) ), with a second-order rate constant ((7.00 ± 0.62) × 10⁴ M⁻¹ s⁻¹) at pH 7.4. We tried to measure the UV–vis absorption spectrum of ${\text{As}}^{-\cdot }$ radical, by oxidizing AsH⁻ with air (³O₂) in the buffer solution at pH 7.4. However, only a decrease in the UV–vis absorption spectrum of AsH⁻ at 266 nm was observed, while the spectrum of ${\text{As}}^{-\cdot }$ radical was not observed at 360 nm (data not shown). As shown in Fig. 3(A), the absorption peak due to ${\text{As}}^{-\cdot }$ radicals was not observed at ~360 nm.

To our knowledge, the UV–vis absorption spectrum of ${\text{PQQH}}^{\cdot }$ radical has not been reported earlier, as ${\text{PQQH}}^{\cdot }$ radical is considered to be unstable. In fact, the absorption which will be due to ${\text{PQQH}}^{\cdot }$ radicals was not observed for the reaction of PQQ with Vit C, as shown in Fig. 3. PQQH₂ and (−)-epicatechin (or (+)-catechin) are the molecules possessing a catechol structure. (−)-Epicatechin and (+)-catechin radicals, which are the transient products due to their AO action, are known to be unstable. Jovanovic et al.^42,43) have measured the absorption spectra of these radicals using pulse radiolysis technique. It has been reported that (−)-epicatechin and (+)-catechin radicals disappear by disproportionation reaction, producing ortho-quinone products of (+)-catechin and (−)-epicatechin.^44,45) Similarly, ${\text{PQQH}}^{\cdot }$ radicals generated by the reaction (7(7) $$\text{PQQ}+{\text{AsH}}^{-}\to {\text{PQQH}}^{\cdot }+{\text{As}}^{-\cdot }$$(7) ) will disappear rapidly by (i) reaction (8(8) $${\text{PQQH}}^{\cdot }+{\text{AsH}}^{-}\to {\text{PQQH}}_2+{\text{As}}^{-\cdot }$$(8) ) and/or (ii) disproportionation reaction (9(9) $${\text{PQQH}}^{\cdot }+{\text{PQQH}}^{\cdot }\to \text{PQQ}+{\text{PQQH}}_2$$(9) ). However, the details of the decay mechanism of ${\text{PQQH}}^{\cdot }$ radicals are not well understood at present.

PQQNa₂ is reduced by Vit C producing PQQH₂, which is further recycled to PQQ by air oxidation in buffer solution at pH 7.4

As described in the Results section, by adding the 0.05 M phosphate-buffered solution (pH 7.4) of Vit C (2 × 5.36 × 10⁻⁴ M) to the solution of PQQNa₂ (2 × 4.45 × 10⁻⁵ M) (1:1 in volume) at 25 °C, the absorption spectrum of PQQNa₂ disappeared rapidly, and finally changed to that of PQQH₂ (see Fig. 3(A) and (B)). The time dependence of the absorbance at 307.5 nm is shown in Fig. 4(A).

The reaction mixture includes PQQNa₂ and Vit C in the buffer solution. If only PQQNa₂ and Vit C coexist in the buffer solution (see reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) )), the total absorbance (${\text{Abs}}_{\text{initial}}^{\text{total}}$) of PQQNa₂ and Vit C at the beginning of the reaction could be expressed as given in Equation (11(11) $$\begin{array}{cc}{\text{Abs}}_{\text{initial}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{initial}}^{{\text{PQQNa}}_2}+{\text{Abs}}_{\text{initial}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQNa}}_2}{[{\text{PQQNa}}_2]}_{\text{initial}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{initial}}\hfill \end{array}$$(11) ).(11) $$\begin{array}{cc}{\text{Abs}}_{\text{initial}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{initial}}^{{\text{PQQNa}}_2}+{\text{Abs}}_{\text{initial}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQNa}}_2}{[{\text{PQQNa}}_2]}_{\text{initial}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{initial}}\hfill \end{array}$$(11)

where ${\mathit{\epsilon }}^{{\text{PQQNa}}_2}$ and ε^{Vit C} are the molar extinction coefficients of PQQNa₂ and Vit C, respectively, at 307.5 nm in the buffer solution (pH 7.4) (see Table 1). [PQQNa₂]_initial and [Vit C]_initial are the initial concentrations of the reactants PQQNa₂ and Vit C, respectively, which were used for reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ) (with low conc. of Vit C), as listed in Table 2. The ${\text{Abs}}_{\text{initial}}^{\text{total}}$ value (0.525) at t = 0 min calculated using Equation (11(11) $$\begin{array}{cc}{\text{Abs}}_{\text{initial}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{initial}}^{{\text{PQQNa}}_2}+{\text{Abs}}_{\text{initial}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQNa}}_2}{[{\text{PQQNa}}_2]}_{\text{initial}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{initial}}\hfill \end{array}$$(11) ) is in good agreement with the observed value (0.544) listed in Table 2. These results suggest that the method of analysis performed in the present study could be considered reliable.

Table 2. Changes in the absorbance of PQQH₂ at 307.5 nm due to the reaction of PQQNa₂ with vitamin C in the buffer solution (pH 7.4) at 25.0 °C under nitrogen atmosphere.

	at t = 0 min		at t = 15 h
	$\begin{array}{c}{\text{Abs}}_{\text{initial}}^{\text{total}}\\ ={\text{Abs}}_{\text{initial}}^{{\text{PQQNa}}_2}+{\text{Abs}}_{\text{initial}}^{\text{Vit C}}\\ =\mathit{\epsilon }[{\text{PQQNa}}_2]+{\mathit{\epsilon }}^{\text{Vit C}}[\text{Vit C}]\end{array}$	${\text{Abs}}_{\text{initial}}^{\text{total}}$	$\begin{array}{c}{\text{Abs}}_{\text{final}}^{\text{total}}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}+{\text{Abs}}_{\text{final}}^{\text{Vit C}}\\ ={\mathit{\epsilon }}^{{\text{PQQH}}_2}[{\text{PQQH}}_2]+{\mathit{\epsilon }}^{\text{Vit C}}[\text{Vit C}]\end{array}$	${\text{Abs}}_{\text{final}}^{\text{total}}$
Reaction 5 (low conc. of Vit C)	= 10,800[4.45 × 10^−5] + 82.6[5.36 × 10^−4]	0.525 (calcd.)	= 37,700[4.45 × 10^−5]+82.6[4.47 × 10^-4]	1.715 (calcd.)
	= 0.481 + 0.044		=1.678 + 0.037
	=0.500^a + 0.044	0.544 (obsd.)	=1.423^b + 0.037	1.460 (obsd.)
			$\begin{array}{c}\text{ratio}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}(\text{obsd}.)/{\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}(\text{calcd}.)\\ =1.423/1.678=0.848\end{array}$
	at t = 0 min		at t = 15 h
Reaction 5 (high conc. of Vit C)	=10,800[4.45 × 10^−5] + 82.6[5.36 × 10^-3]	0.924 (calcd.)	=37,700[4.45 × 10^−5] + 82.6[5.27 × 10^−3]	2.120 (calcd.)
	=0.481 + 0.443		=1.678 + 0.435
	=0.935^a + 0.443	1.378 (obsd.)	=1.867^b + 0.435	2.302 (obsd.)
			$\begin{array}{c}\text{ratio}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}(\text{obsd}.)/{\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}(\text{calcd}.)\\ =1.867/1.678=1.11\end{array}$

^a ${\text{Abs}}_{\text{initial}}^{{\text{PQQH}}_2}$ (obsd.) value was obtained by subtracting the contribution of ${\text{Abs}}_{\text{initial}}^{\text{Vit C}}$ (calcd.) from ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.).

^b ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (obsd.) value was obtained by subtracting the contribution of ${\text{Abs}}_{\text{final}}^{\text{Vit C}}$ (calcd.) from ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.).

By reacting PQQNa₂ with Vit C under nitrogen atmosphere, PQQNa₂ is reduced and produces PQQH₂ (see Fig. 3(A) and (B)). As described in a previous section, ${\text{As}}^{-\cdot }$ and ${\text{PQQH}}^{\cdot }$ radicals are unstable in the buffer solution at pH 7.4 and do not contribute to the change in the absorption spectra observed due to the reaction between PQQNa₂ and Vit C. Therefore, if the reduction reaction of PQQNa₂ with Vit C is completed at t = 15 h (see Fig. 4(A)), the ${\text{Abs}}_{\text{final}}^{\text{total}}$ value of PQQH₂ and Vit C in solution could be calculated using Equation (12(12) $$\begin{array}{cc}{\text{Abs}}_{\text{final}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}+{\text{Abs}}_{\text{final}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQH}}_2}{[{\text{PQQH}}_2]}_{\text{final}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{final}}\hfill \end{array}$$(12) ) as follows:(12) $$\begin{array}{cc}{\text{Abs}}_{\text{final}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}+{\text{Abs}}_{\text{final}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQH}}_2}{[{\text{PQQH}}_2]}_{\text{final}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{final}}\hfill \end{array}$$(12)

where [PQQH₂]_final is the final concentration of PQQH₂ produced as a result of the reduction of Vit C. In order to reduce one molecule of PQQ to one molecule of PQQH₂, two molecules of Vit C are required (see reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) )). Consequently, [Vit C] changes from [Vit C]_initial (5.36 × 10⁻⁴ M) at t = 0 min to [Vit C]_final (5.36 × 10⁻⁴ – 2 × 4.45 × 10⁻⁵ = 4.47 × 10⁻⁴ M) at t = 15 h, where [PQQNa₂]_initial is equal to 4.45 × 10⁻⁵ M.

The values of ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (calcd.), ${\text{Abs}}_{\text{final}}^{\text{Vit C}}$ (calcd.), and ${\text{Abs}}_{\text{final}}^{\text{total}}$ (calcd.) at t = 15 h were found to be 1.678, 0.037, and 1.715, respectively, according to Equation (12(12) $$\begin{array}{cc}{\text{Abs}}_{\text{final}}^{\text{total}}\hfill & ={\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}+{\text{Abs}}_{\text{final}}^{\text{Vit C}}\hfill \\ \hfill & ={\mathit{\epsilon }}^{{\text{PQQH}}_2}{[{\text{PQQH}}_2]}_{\text{final}}+{\mathit{\epsilon }}^{\text{Vit C}}{[\text{Vit C}]}_{\text{final}}\hfill \end{array}$$(12) ) and are listed in Table 2. The contribution of ${\text{Abs}}_{\text{final}}^{\text{Vit C}}$ (calcd.) to ${\text{Abs}}_{\text{final}}^{\text{total}}$ (calcd.) is small (~2.2%) and seems to be almost negligible. The value of ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (obsd.), i.e., 1.423 was estimated by subtracting the contribution of Vit C (${\text{Abs}}_{\text{final}}^{\text{Vit C}}$ (calcd.) (0.037)) from the total ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.) (1.460). A comparison of the ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (obsd.) (1.423) with ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (calcd.) (1.678) suggests that the former value is 0.85 times smaller than the latter. The ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.) value (1.460) (or ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (obsd.) (1.423)) at t = 15 h increases gradually and seems to approach the ${\text{Abs}}_{\text{final}}^{\text{total}}$ (calcd.) (1.715) (or ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (calcd.) (1.678)) values, as shown in Fig. 4(A).

On the other hand, by introducing air into the reaction mixture containing PQQH₂ and Vit C (with residual PQQ in mixture), rapid change in the absorption spectrum was observed due to the oxidation of PQQH₂ to PQQ (Fig. 6). As shown in Fig. 4(B), the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.) value (1.420) decreases gradually from t = 0 to 120 min and slowly approaches to a constant value of 0.544 (see Table 3). This value is very similar to the values of ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.) (0.544) and ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (calcd.) (0.525) that are obtained at t = 0 min for the reaction between PQQNa₂ and Vit C (Table 2). As listed in Table 3, the ratio of ${\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}$ (obsd.) to ${\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}$ (calcd.) (=0.507/0.481) is 1.05. This result indicates that PQQH₂ is completely recycled to the original PQQ due to air oxidation, as shown in Fig. 7.

Table 3. Changes in the absorbance of PQQH₂ at 307.5 nm due to the reaction of PQQH₂ with air (molecular oxygen) in the buffer solution (pH 7.4) at 25.0 °C.

	at t = 0 min	at t = 15 h
	${\text{Abs}}_{\text{initial}}^{\text{total}}$	$\begin{array}{c}{\text{Abs}}_{\text{final}}^{\text{total}}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}+{\text{Abs}}_{\text{final}}^{\text{Vit C}}\\ ={\mathit{\epsilon }}^{{\text{PQQNa}}_2}[{\text{PQQNa}}_2]+{\mathit{\epsilon }}^{\text{Vit C}}[\text{Vit C}]\end{array}$	${\text{Abs}}_{\text{final}}^{\text{total}}$
Reaction 5 (low conc. of Vit C)		=10,800[4.45 × 10^−5] + 82.6[4.47 × 10^−4]	0.518 (calcd.)
		= 0.481 + 0.037
	1.420 (obsd.)	=0.507^a + 0.037	0.544 (obsd.)
		$\begin{array}{c}\text{ratio}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}(\text{obsd}.)/{\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}(\text{calcd}.)\\ =0.507/0.481=1.05\end{array}$
	at t = 0 min	at t = 38 h
Reaction 5 (high conc. of Vit C)		=10,800[4.45 × 10^−5] + 82.6[5.27 × 10^−3]	0.916 (calcd.)
		=0.481 + 0.435
	2.305 (obsd.)	=1.030^a + 0.435	1.465 (obsd.)
		$\begin{array}{c}\text{ratio}\\ ={\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}(\text{obsd}.)/{\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}(\text{calcd}.)\\ =1.030/0.481=2.14\end{array}$

^a ${\text{Abs}}_{\text{final}}^{{\text{PQQH}}_2}$ (obsd.) value was obtained by subtracting the contribution of ${\text{Abs}}_{\text{final}}^{\text{Vit C}}$ (calcd.) from ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.).

As described in the Results section, the reaction between PQQNa₂ and Vit C was performed by keeping the concentration of PQQNa₂ ([4.45 × 10⁻⁵ M]) constant and using a 10 times higher concentration of Vit C ([5.36 × 10⁻³ M]) (see reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ) with high conc. of Vit C) (Table 2 and Fig. 5(A) and (B)). The initial and final values of Abs^PQQH2 (calcd.), Abs^{Vit C} (calcd.), and Abs^total (calcd.) are listed in Table 2, together with the values of Abs^PQQH2 (obsd.), Abs^{Vit C} (obsd.), and Abs^total (obsd.) observed.

As described above, the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.) value (0.544) observed for the reactants showed a good agreement with the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (calcd.) value (0.525), when [Vit C]_initial was low (5.36 × 10⁻⁴ M). However, when a 10 times higher concentration of Vit C ([Vit C]_initial = 5.36 × 10⁻³ M) was used for the reaction, the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.) value (1.378) at t = 0 min was 1.49 times larger than the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (calcd.) one (0.924). This is because it takes about 3 min (dead time) before (i) mixing the two buffer solutions containing PQQNa₂ and Vit C, and (ii) in recording the first UV–vis spectrum. As shown in Fig. 5(A), the rate of the increase in the absorbance of PQQH₂ for the reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ) (high conc. of Vit C) (indicated by ●) is much faster than that for the reaction (5(5) $$\text{PQQ}+2\text{Vit C}(\text{or}2{\text{AsH}}^{-})\to {\text{PQQH}}_2+2{\text{Vit C}}^{\cdot }(\text{or}2{\text{As}}^{-\cdot })$$(5) ) (low conc. of Vit C) (indicated by ○). Consequently, the increase in the ${\text{Abs}}_{\text{initial}}^{\text{total}}$ (obsd.) value from 0.924 to 1.378 at t = 0 min may be explained by considering that the reaction occurred during the dead time.

As shown in Fig. 5(A), for the reaction of PQQNa₂ with Vit C ([Vit C] = 5.36 × 10⁻³ M), the Abs^total (obsd.) value (indicated by ●) increases rapidly from 1.378 at t = 0 min and shows a constant value (~2.10) at t = 7–12 min, suggesting that the reduction reaction is completed at around t = 7 min. In fact, the Abs^total (obsd.) value (~2.10) is similar to the Abs^total (calcd.) (2.120) (Table 2). However, the Abs^total (obsd.) value increases gradually from ~2.10 to 2.302 at t = 15 h. As listed in Table 2, the ratio of ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.) to ${\text{Abs}}_{\text{final}}^{\text{total}}$ (calcd.) (=2.302/2.120) is 1.09. The reason for such an increase of the ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.) value is not clearly understood at present.

Furthermore, by introducing air into the reaction mixture, the Abs^total (obsd.) value (indicated by ●) decreases slowly (see Fig. 5(B)). At t = 38 h in the presence of air, ${\text{Abs}}_{\text{final}}^{\text{total}}$ (obsd.) value (1.465) is 1.59 times larger than ${\text{Abs}}_{\text{final}}^{\text{total}}$ (calcd.) (0.923). The high concentration of Vit C included in the solution could suppress the oxidation of PQQH₂ to PQQ. A notable difference in the time dependence of Abs^total (obsd.) value is clear from the plots shown in Fig. 5(B). As listed in Table 3, the ratio of ${\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}$ (obsd.) to ${\text{Abs}}_{\text{final}}^{{\text{PQQNa}}_2}$ (calcd.) (1.030/0.481) is 2.14, indicating that about one half of PQQH₂ is not oxidized and is left unconsumed in the solution. Vit C, which coexists in the reaction mixture, functions as an AO and prevents the oxidation of PQQH₂ to PQQ.

PQQ is reduced to PQQH₂ by Vit C, which may function as an AO in biological systems

As described in the Introduction section, PQQ and Vit C coexist in many foods^{2,19–21,31)} and human and rat tissues.^{22,23,32–34)} High concentrations of Vit C are also present in human plasma³²⁾ and coexist with PQQ.²²⁾ The results obtained in the present study suggest that PQQ is reduced to PQQH₂ by Vit C, which functions as an AO in many biological systems. This is because it has been reported that PQQH₂ shows very high free-radical scavenging and singlet-oxygen quenching activities in buffer solution at pH 7.4.^26,29) Furthermore, the results obtained in the present study suggest that PQQ and PQQH₂ may be recycled, for example, in the human plasma, as shown in Fig. 7.

In a recent study, the measurement of ${\text{ArO}}^{\cdot }$-scavenging rate constants (k_s) for AOs (such as PQQH₂, α-TocH, UQ₁₀H₂, and three catechins) were performed in dimethyl sulfoxide (DMSO) solution using stopped-flow spectrophotometry (see reaction (2$$k_s$$)).⁴⁶⁾ In this study, the values for k_s were not only measured for each of the AOs, but also for the mixture of two AOs, i.e., (i) α-TocH and PQQH₂ and (ii) α-TocH and UQ₁₀H₂. A notable synergistic effect was observed which suggested that the k_s values increase by 1.72, 2.42, and 2.50 times for α-TocH, PQQH₂, and UQ₁₀H₂, respectively, for the solutions including two kinds of AOs. The measurements of the regeneration rate constants of α-tocopheroxyl radical ($\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }$) to α-TocH in the presence of PQQH₂ and UQ₁₀H₂ were performed in DMSO using a double-mixing stopped-flow spectrophotometry technique (reaction (13(13) $$\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }+{\text{PQQH}}_2\to \mathit{\alpha }\text{-}\text{TocH}+{\text{PQQH}}^{\cdot }$$(13) )). The second-order rate constant (k_r) (1.08 × 10⁵ M⁻¹ s⁻¹) obtained for PQQH₂ was found to be 3.0 times larger than that (3.57 × 10⁴) for UQ₁₀H₂. This result indicates that the pro-oxidant effect of $\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }$ is suppressed by the coexistence of PQQH₂ or UQ₁₀H₂.$$k_r$$(13) $$\mathit{\alpha }\text{-}{\text{Toc}}^{\cdot }+{\text{PQQH}}_2\to \mathit{\alpha }\text{-}\text{TocH}+{\text{PQQH}}^{\cdot }$$(13)

As reported in the previous studies, PQQ coexists with α-TocH in foods and biological systems (such as plasma, blood, and various tissues).^2,19,20,22) Consequently, the above-mentioned synergistic effects, i.e., an increase in the free-radical scavenging rate and the suppression of the pro-oxidant reaction may also function in foods and biological systems. Recently, in light of its beneficial physiological effects, PQQ has been identified as a potential candidate for its use in the dietary supplements.⁴⁷⁾ The results obtained in the present study suggest that the use of supplements including PQQNa₂, Vit C, and α-TocH at the same time is preferable in maintaining a good health.

Author contributions

K. Mukai designed research and wrote the manuscript. A. Ouchi performed experiment and analyzed the data. S. Nagaoka helped to draft the manuscript and supervised the research. K. Ikemoto and M. Nakano prepared sodium salt of PQQ and provided valuable advice.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was partly supported by the Challenging Exploratory Research from the Japan Society for the Promotion of Science [grant number 24658123].