Biocatalysts are widely used in industry, but few examples of the use of oxidoreductases, in which enzymatic function often requires electrons, have been reported. NADPH is a cofactor that supplies an electron to oxidoreductases, but is consequently inactivated and no longer able to act as an electron donor. NADP+ can not receive electrons from electrodes through straightforward electrochemistry owing to its complicated three-dimensional structure. This study reports that bipyridines effectively mediate electron transfer between an electrode and NADP+, allowing them to serve as electron mediators for NADPH production. Using bipyridines, quinones, and anilines, which have negative oxidation–reduction potentials, an electrochemical investigation was conducted into whether electrons were transferred to NADP+. Only bipyridines with a reduction potential near -1.0 V exhibited electron transfer. Furthermore, the NADPH production level was measured using spectroscopy. NADPH was efficiently produced using bipyridines, such as methyl viologen and ethyl viologen, in which the bipyridyl 1- and 1’-positions bear small substituents. However, methyl viologen caused a dehydrogenation reaction of NADPH, making it unsuitable as an electron mediator for NADPH production. The dehydrogenation reaction did not occur using ethyl viologen. These results indicated that NADP+ can be reduced more effectively using substituents that prevent a dehydrogenation reaction at the bipyridyl 1- and 1’-positions while maintaining the reducing power.
Citation: Wayama F, Hatsugai N, Okumura Y (2022) Bipyridines mediate electron transfer from an electrode to nicotinamide adenine dinucleotide phosphate. PLoS ONE 17(6): e0269693. https://doi.org/10.1371/journal.pone.0269693
Editor: Roswanira Abdul Wahab, Universiti Teknologi Malaysia, MALAYSIA
Received: November 5, 2021; Accepted: May 25, 2022; Published: June 16, 2022
Copyright: © 2022 Wayama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the article.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
To achieve sustainability, materials synthesis should have a low environmental impact and be efficient. In this context, biotechnology is a key technology for future economic development and pertinent dynamic growth opportunities. Biocatalysts, such as enzymes and microorganisms, are common in material production owing to their diminished effects on the environment compared with chemical catalysts [1–5]. Unlike chemical conversion, biochemical conversion does not require high temperatures, high pressures, or extreme pH values. Biocatalysts also have high specificity for their substrates. Examples of industrial biocatalysts and their applications are as follows [for reviews, see refs. 6 and 7]: (i) Lipase, cellulase, amylase, and protease as detergent enzymes; (ii) cellulase and protease in fiber processing; (iii) lipase in papermaking; (iv) xylanase, in pulp bleaching; and (v) lipase and amylase in food production. However, there are few examples of oxidoreductase use in industry. This is because some of oxidoreductases require a cofactor to function, which supplies electrons to reactivate oxidoreductase that has been inactivated by reaction with the substrate. The repeated reactivation of oxidoreductase that allows the reaction to proceed with a small amount of oxidoreductase, enables the in vitro utilization of in vivo reaction and material production with a lower environmental impact.
Some oxidoreductases require NADPH as a cofactor, which function as electron carriers [8–10]. However, when oxidoreductases use a cofactor in vitro, in vivo function is difficult to mimic because no substance is available to oxidize/reduce the cofactor after acting on the enzyme. Also, NADPH is too expensive to use on a large scale. Therefore, NADP+ reduction (i.e., NADPH recycling) is crucial for the utilization of NADPH-dependent enzymes [9–13]. Electrochemistry is often used to transfer electrons for various substances. However, because the structure of NADP is complex, electron transport generally requires an enzyme that can exchange NADP+ to NADPH [9–13]. The direct exchange of electrons with an electrode is difficult.
Electron mediators can mediate electron transfer between an electrode and enzyme [9,14–16]. Electron mediators have a reversible redox potential, and readily transfer electrons to and from the electrode, even after oxidizing/reducing the target molecule, allowing the mediator to continue acting on the target molecule (Fig 1). In this study, we selected the electronic mediators that directly transfer electrons efficiently and continuously from an electrode to NADP+. Bipyridines were shown to effectively transport electrons from working electrode and produce NADPH in conjunction with proton supply from the counter electrode. Our results provide valuable insights for industrial biocatalysts using oxidoreductases.
Nicotinamide adenine dinucleotide phosphate (NADP+) was obtained from Wako. NADP+ solutions were prepared in phosphate-buffered saline (PBS; pH 7.4). 2-Hydroxy-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 2,6-dimethoxy-1,4-benzoquinone, 1,4-naphthoquinone, 1,4-benzoquinone, 2,3,5,6-tetramethyl-1,4-phenylenediamine, N,N,N’,N’-tetramethyl-1,4-phenylenediamine, 1,1’-diphenyl-4,4’-bipyridinium, 1,1’-bis(2,4-dinitrophenyl)-4,4’-bipyridinium, 1,1’-difluoro-2,2’-bipyridinium, and 3,3’-Bipyrid were obtained from Tokyo Chemical Industry Co., Ltd. 4,4’-Dipyridyl, methyl viologen, ethyl viologen, 1-heptyl-4-(4-pyridyl) pyridinium, 1,1’-diheptyl-4,4’-bipyridinium, benzyl viologen, and 2,2’-dipyridyl were purchased from Sigma Aldrich. All mediators were dissolved in PBS and adjusted to 1 mM.
All electrochemical measurements were performed using an electrochemical analyzer (BAS; ALS Model 612E). Phosphate buffer (pH 7.4) was used as the sample solution, from which oxygen was removed using flowing argon for 15 min before measurement. Electron transport from the electron mediator to NADP+ was measured using cyclic voltammetry. At room temperature (25°C), a voltage was applied in the range of 1.0 to -1.5 V at a sweep rate of 50 mV/s. The electron mediator concentration in the sample (6 mL) was 1 mM, and the NADP+ concentration was 0, 1, or 2 mM. The working, counter, and reference electrodes were glassy carbon, Pt, and Ag/AgCl, respectively.
Absorption spectrum measurement
To investigate NADP+ reduction and NADPH production via the electron mediator, the absorption spectrum of the mixed solution of the electron mediator and NADP+ was measured while applying a voltage. To confirm NADPH production, sample (1 mL) was placed in a 1-cm square cuvette and a voltage of -0.75 V or -1.05 V was applied for 30 min. The working, counter, and reference electrodes were gold mesh, Pt, and Ag/AgCl (BAS), respectively. An electrochemical analyzer (BAS; ALS Model 612E) was used for voltage application. Regarding the absorption spectrum, a change in absorbance at 340 nm, which is the absorption wavelength of NADPH, was observed using a GeneQuant 1300 spectrophotometer.
Results and discussion
Redox potential of each low-molecular-weight compound
The size, redox potential, and chemical characteristics of electron mediators can influence their electron transport properties. Low-molecular-weight compounds smaller than NADP+, namely, bipyridines, quinones, and anilines, were selected as candidate mediators. Reduction potentials were determined by cyclic voltammetry. Table 1 shows the measured reduction potentials of each electron mediator candidate. The reduction potential of NADP+ is -520 mV (vs. Ag/AgCl) . Therefore, an electron mediator to reduce NADP+ needs to have a reduction peak at a potential lower than -520 mV. Among the low-molecular-weight compounds shown in Table 1, 2-hydroxy-1,4-naphthoquinone, 4,4’-dipyridyl, methyl viologen, ethyl viologen, 1-heptyl-4-(4-pyridyl) pyridinium, 1,1’-diheptyl-4,4’-bipyridinium, and benzyl viologen were selected as promising electronic mediators.
Electron transfer from electrode to NADP+ via electron mediator
To evaluate the ability of the electron mediator candidates to transport electrons from an electrode to NADP+, their redox current peaks were measured in the presence and absence of NADP+ by cyclic voltammetry. In the absence of the electronic mediator, NADP reduced only slightly even when a large voltage was applied [Fig 2(A)]. The reduction current peak of 4,4’-dipyridyl increased in the presence of NADP+ [Fig 2(B)]. Methyl viologen and ethyl viologen had two redox potentials. This indicates that methyl viologen and ethyl viologen are oxidized and reduced in two steps. Methylviologen shows one midpoint potential much larger than the other. There is a possibility of this phenomenon because one-electron transfer and two-electron transfer are occurring at the same time around -1.0V . At a reduction potential near -750 mV, no change in the reduction current peak was observed in the presence of NADP+. Meanwhile, at a reduction potential near -1.05 V, the reduction current peak increased in the presence of NADP+ [Fig 2(C) and 2(D)]. Methyl viologen exhibited the largest increase in reduction current, indicating that it would transport electrons most efficiently among the tested candidates. Furthermore, a concurrent decrease in the oxidation current was observed. The reduction and oxidation current peaks were both NADP+ concentration-dependent. We proposed that this was due to the reduced electron mediator immediately transforming back into an oxidant owing to the presence of NADP+ during the sweep toward a negative potential. The electron mediators transforming back into an oxidant are also reduced in sequence, such that the reduction current increased. Furthermore, electron mediator remained an oxidant when the potential direction was switched from negative to positive, such that the oxidization current decreased. Therefore, three electron mediators, namely, 4,4’-dipyridyl, methyl viologen, and ethyl viologen, were able to transport electrons from the electrode to NADP+. The other four electronic mediators showed no change in the voltammogram with or without NADP+ [Fig 2(E)–2(G)].
Black line: the absence of NADP+. Red line: the presence of 1 mM NADP+. Blue line: the presence of 2mM NADP+. Potential scan rate, 50 mV/s. (A) Control (none), (B) 4,4′-Dipyridyl, (C) Methyl viologen, (D) Ethyl viologen, (E) 1-Heptyl-4-(4-pyridyl) pyridinium (F) 1,1′-Diheptyl-4,4′-bipyridinium, (G) Benzyl viologen. These experiments were repeated three times with similar results.
NADPH production by electron transport via electron mediator
Cyclic voltammetry showed that methyl viologen and ethyl viologen had two redox peaks, indicating that they were one- and two-electron reducers. When reduced by one electron to become a radical cation, these compounds exhibited an absorption peak near 395 nm and developed a blue color. When reduced by two electrons, this absorption was not present and the solution was colorless.
To evaluate NADPH production, which exhibits an absorption peak near 340 nm, the absorbance of solutions containing an electron mediator and NADP+ was measured while applying a voltage. When a voltage of -0.75 V was applied (first reduction potential) to a mixed solution of methyl viologen and NADP+, the solution turned blue after 30 min, and absorption near 395 nm was observed [Fig 3(A)]. The absorbance at 395 nm continued to increase monotonically with increasing time under the applied voltage. No change in absorbance was observed near 340 nm. However, when a voltage of -1.05 V was applied (second reduction potential), the absorbance near 395 nm increased immediately and then decreased after 30 s. As the absorbance near 395 nm decreased, the absorbance near 340 nm increased [Fig 3(B)]. These increases and decreases in absorption were detected similarly at 620 nm.
(A) -0.75 V. (B) -1.05 V. Voltage application time 0 s—30 min. These experiments were repeated three times with similar results.
Similarly, for ethyl viologen, the one-electron reducer was not able to reduce NADP+, while the two-electron reducer achieved reduction of NADP+. Furthermore, NADPH generation was confirmed using 4,4’-dipyridyl, which has a reduction potential near -1.0 V. Therefore, NADPH (reduced NADP+) can be produced using an electron mediator with a reduction potential near -1.0 V (Fig 4). Furthermore, 4,4’-dipyridyl produced the smallest quantity of NADPH. We proposed that this was due to the reduction potential of 4,4’-dipyridyl being lower than those of methyl viologen and ethyl viologen (Fig 4). In addition, 4,4’-dipyridyl does not have a substituent at the 4,4’position, so it may function as a base and inhibit the movement of protons.
On the left is the absorption spectrum from 300 nm to 480 nm. On the right is a plot of absorbance at 340 nm and 390 nm for each hour. (A) Methyl viologen, (B) Ethyl viologen, (C) 4,4′-Dipyridyl. These experiments were repeated three times with similar results.
Among the mediator candidates, methyl viologen most effectively transported electrons to NADP+, while ethyl viologen produced a larger quantity of NADPH. Methylviologen is known to transfer electrons from NADPH in vivo and oxidize NADPH to NADP+ . For a mixed solution of methyl viologen and NADPH, the measured absorbance at 340 nm (attributable to NADPH) decreased over time [Fig 5(A)]. However, the absorbance of a mixed solution of ethyl viologen and NADPH showed no change [Fig 5(B)]. Therefore, the difference in NADPH amount afforded by methyl viologen compared with ethyl viologen was due to the fact that methylviologen oxidizes NADPH.
NADP+ was clarified to be reduced electrochemically by an electron mediator with a reduction potential near -1.0 V. Bipyridyl was an effective electronic mediator, while methyl viologen was not appropriate due to causing NADPH to undergo a dehydrogenation reaction. Furthermore, when substituents at the bipyridyl 1- and 1’-positions were large, the reduction potential shifted higher, preventing the reduction of NADP+. Therefore, to produce NADPH, bipyridyl compounds bearing relatively small functional groups, such as ethyl groups, are appropriate for use as electronic mediators.
- 1. Bornscheuer U. T.; Huisman G. W.; Kazlauskas R. J.; Lutz S.; Moore J. C.; Robins K. Engineering the third wave of biocatalysis. Nature 2012, 485(7397), 185–194. pmid:22575958
- 2. Wenda S.; Illner S.; Mell A.; Kragl U. Industrial biotechnology—the future of green chemistry? Green Chemistry 2011, 13(11), 3007–3047.
- 3. Choi J. M.; Han S. S.; Kim H. S. Industrial applications of enzyme biocatalysis: Current status and future aspects. Biotechnology Advances 2015, 33(7), 1443–1454. pmid:25747291
- 4. Schmid A.; Dordick J. S.; Hauer B.; Kiener A.; Wubbolts M.; Witholt B. Industrial biocatalysis today and tomorrow. Nature 2001, 409(6817), 258–268. pmid:11196655
- 5. Wohlgemuth R. Biocatalysis—key to sustainable industrial chemistry. Current Opinion in Biotechnology 2010, 21(6), 713–724. pmid:21030244
- 6. Kirk O.; Borchert T. V.; Fuglsang C. C. Industrial enzyme applications. Current Opinion in Biotechnology 2002, 13(4), 345–351. pmid:12323357
- 7. Bajpai P. Application of enzymes in the pulp and paper industry. Biotechnology Progress 1999, 15(2), 147–157. pmid:10194388
- 8. Møller I. M.; Rasmusson A. G. The role of NADP in the mitochondrial matrix. Trends in Plant Science 1998, 3(1), 21–27.
- 9. Dinh T. H.; Lee S. C.; Hou C. Y.; Won K. Diaphorase-viologen conjugates as bioelectrocatalysts for NADH regeneration. Journal of The Electrochemical Society 2016, 163(6), H440.
- 10. Wu H.; Tian C.; Song X.; Liu C.; Yang D.; Jiang Z. Methods for the regeneration of nicotinamide coenzymes. Green Chemistry 2013, 15(7), 1773–1789.
- 11. Weckbecker A.; Gröger H.; Hummel W. Regeneration of nicotinamide coenzymes: principles and applications for the synthesis of chiral compounds. Biosystems Engineering I 2010, 195–242. pmid:20182929
- 12. Siritanaratkul B.; Megarity C. F.; Roberts T. G.; Samuels T. O.; Winkler M.; Warner J. H., et al. Transfer of photosynthetic NADP+/NADPH recycling activity to a porous metal oxide for highly specific, electrochemically-driven organic synthesis. Chemical Science 2017, 8(6), 4579–4586. pmid:30155220
- 13. Reeve H. A.; Lauterbach L.; Lenz O.; Vincent K. A. Enzyme-modified particles for selective biocatalytic hydrogenation by hydrogen-driven NADH recycling. ChemCatChem 2015, 7(21), 3480. pmid:26613009
- 14. Komoschinski J.; Steckhan E. Efficient indirect electrochemical in situ regeneration of NAD+ and NADP+ for enzymatic oxidations using iron bipyridine and phenanthroline complexes as redox catalysts. Tetrahedron letters 1988, 29(27), 3299–3300.
- 15. Bes M. T.; de Lacey A. L.; Fernandez V. M.; Gomez-Moreno C. Electron transfer between viologen derivatives and the flavoprotein ferredoxin-NADP+ reductase. Bioelectrochemistry and bioenergetics 1995, 38(1), 179–184.
- 16. Kim S.; Yun S. E.; Kang C. Electrochemical evaluation of the reaction rate between methyl viologen mediator and diaphorase enzyme for the electrocatalytic reduction of NAD+ and digital simulation for its voltammetric responses. Journal of Electroanalytical Chemistry 1999, 465(2), 153–159.
- 17. Chen X.; Cao Y.; Li F.; Tian Y.; Song H. Enzyme-Assisted Microbial Electrosynthesis of Poly(3-hydroxybutyrate) via CO2 Bioreduction by Engineered Ralstonia eutropha. ACS Catalysis 2018, 8(5), 4429–4437.
- 18. Cimino P.; Raucci U.; Donati G.; Chiariello M. G.; Schiazza M.; Coppola F.; et al. On the different strength of photoacids. Theoretical Chemistry Accounts 2016, 135(5), 1–12.
- 19. Shoichi M. Paraquat, an active-oxygen producing herbicide. Tanpakushitsu Kakusan Koso 1988, Dec;33(16):2790–4. pmid:2855145