Phenothiazine Dyes Induce NADH Photooxidation through Electron Transfer: Kinetics and the Effect of Copper Ions.

Phenothiazine dyes, methylene blue, new methylene blue, azure A, and azure B, photosensitized the oxidation of nicotinamide adenine dinucleotide (NADH), an important coenzyme in the living cells, through electron transfer. The reduced forms of these phenothiazine dyes, which were produced through electron extraction from NADH, underwent reoxidation to the original cationic forms, leading to the construction of a photoredox cycle. This reoxidation process was the rate-determining step in the photoredox cycle. The electron extraction from NADH using phenothiazine dyes can trigger the chain reaction of the NADH oxidation. Copper ions enhanced the photoredox cycle through reoxidation of the reduced forms of phenothiazine dyes. New methylene blue demonstrated the highest photooxidative activity in this experiment due to the fast reoxidation process. Electron-transfer-mediated oxidation and the role of endogenous metal ions may be important elements in the photosterilization mechanism.

Introduction

Phenothiazine dyes, such as methylene blue (MB; Figure ), have been applied as agents for antimicrobial photodynamic therapy (aPDT), which is one of the most important medicinal applications of dyes.[1−5] Specifically, aPDT is an advantageous method to sterilize multidrug-resistant bacteria.[6,7] MB and its derivatives (Figure ) can absorb long-wavelength visible light (>650 nm).[3,8] Because long-wavelength visible light can penetrate deeply into biomaterials, including human tissue,[9,10] it is important for photobiological and photomedicinal effects. For example, treatments for periodontal disease,[11,12] dental caries,[13,14] and bone infection[15] are important applications of aPDT using phenothiazine dyes with a red light. Furthermore, viral inactivation photosensitized by phenothiazine dyes has been studied.[16,17] These phenothiazine dyes can photosensitize singlet oxygen (1O2) production with a relatively large quantum yield (ΦΔ) in a solution.[18−20] Therefore, 1O2 has been considered an important reactive species for antimicrobial effects using phenothiazine dyes.[3,6,19] However, biological environments, including the biofilms of microbes, are under hypoxic conditions.[21] Recently, we reported that these phenothiazine dyes oxidize protein through electron transfer under photoirradiation.[22] Furthermore, an analogue compound of MB, Nile blue, also induces DNA oxidation through photoinduced electron transfer.[23] Because the electron-transfer-mediated biomolecule damage does not require an oxygen molecule in the presence of other appropriate oxidative agents such as metal ions, this mechanism may play an important role in photosterilization under hypoxic conditions. In biomolecules, nicotinamide adenine dinucleotide (reduced form; NADH) is easily oxidized by the electron-transfer mechanism, and the formed radical (NAD•) triggers a chain reaction, leading to the acceleration of NADH decomposition and the secondary production of reactive oxygen species.[24] NADH is an important coenzyme and reductant molecule in living cells.[25,26] Thus, we examined the NADH oxidation photosensitized by phenothiazine dyes using MB and its derivatives (azure A (AZA), azure B (AZB), and new methylene blue (NMB); Figure ) in this study. Specifically, the kinetic analysis was performed to investigate the mechanism of the photosensitized reaction.

Figure 1

Structures of the phenothiazine dyes used in this study.

Results and Discussion

Photooxidation of NADH by Phenothiazine Dyes

The typical absorption of NADH at around 340 nm was decreased by photoirradiation with phenothiazine dyes (Figure ). NADH is oxidized to NAD• and the oxidized form (NAD+) through electron transfer or reaction with reactive oxygen species, leading to the diminishing of the typical absorption. The absorption spectra of the phenothiazine dyes were also decreased during this photosensitized reaction. Figure shows the case of MB. The reduction of MB produces the colorless radical form (MB•), or leucomethylene blue (LMB),[27−31] resulting in the diminishing of absorption at around 650 nm. The absorption spectra of these phenothiazine dyes recovered within several minutes under dark conditions. These results suggest that phenothiazine dyes oxidize NADH through electron extraction and that the reduced forms of phenothiazine dyes are reoxidized by oxygen to produce their corresponding cationic forms and superoxide (O2•–).[30,31] Since the recovery of absorption spectra was observed under a dark condition, the photosensitized reaction is faster than the reoxidation process. These processes are summarized using the following equations.orwhere MB+ is the cationic form of MB and MB+* is its photoexcited state.

Figure 2

Absorption spectra of photoirradiated MB and NADH. The sample solution containing 5 μM MB and 100 μM NADH in a 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an light-emitting diode (LED) (λmax = 659 nm, 0.5 mW cm–2). (A) Absorption spectra before and after photoirradiation for 20 min. (B) Time profile of the absorption spectrum in the dark after photoirradiation.

Time Profile of the NADH Photooxidation and Scavenger Effects

The time profiles of NADH oxidation photosensitized by phenothiazine dyes are shown in Figure . Sodium azide (NaN3), a physical scavenger of 1O2,[32] barely inhibited the NADH photooxidation. Furthermore, potassium iodide (KI), a triplet quencher,[33] did not show an inhibitory effect on this photooxidation (Supporting Information). These results suggest that neither the triplet excited (T1) state of these phenothiazine dyes nor 1O2 are responsible for NADH oxidation. However, it is generally accepted that the T1 states of MB and phenothiazine dyes induce oxidative electron transfer from organic molecules.[34,35] KI would serve as a reductant for the phenothiazine dye T1 states and may lead to the formation of O2•– and H2O2.[36] The generation of these secondary reactive oxygen species could enhance NADH oxidation and might offset the inhibitory effect of KI. On the other hand, it has also been reported that the singlet excited (S1) state of MB can oxidize organic compounds through electron transfer.[34,37] Although the possibility of a T1 state-mediated mechanism could not be excluded, the following processes are proposed to explain the observed results. The photosensitized NADH oxidation by phenothiazine dyes can be explained by the electron extraction from NADH to the S1 state of phenothiazine dyes (Dye+*) as followswhere Dye• is the reduced radical form of phenothiazine dyes. The Gibbs free energy (ΔG) of the electron transfer from NADH to the S1 states of these phenothiazine dyes is negative (Table ), supporting this mechanism from the thermodynamic point of view.

Figure 3

Time profile of the NADH decomposed by the photosensitized reaction of phenothiazine dyes. The sample solution containing 5 μM phenothiazine dye (MB, AZA, AZB, or NMB) and 100 μM NADH with or without NaN3 in a 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an LED (λmax= 659 nm, 0.5 mW cm–2).

Table 1

Photochemical and Redox Parameters of Phenothiazine Dyes and the Gibbs Energy of Electron Transfera

dyes	Flmax (nm)	ES1 (eV)	Ered (V) vs SCE	ΔG (eV)
MB	680	1.82	–0.22a	–1.23
AZA	641	1.93	–0.26b	–1.30
AZB	668	1.86	–0.27b	–1.22
NMB	648	1.91	–0.29c	–1.25

Flmax: fluorescence maximum wavelength; ES1: S1 state energy of phenothiazine dyes; Ered: redox potential of one-electron reduction; SCE: saturated calomel electrode; a, b, and c: The values of Ered for a,[38] b,[39] and c[40] are according to the corresponding literatures; the fluorescence spectra of 10 μM dyes (MB, AZA, AZB, or NMB) were measured in a 10 mM sodium phosphate buffer (pH 7.6). The ΔG values were calculated using these values (Supporting Information).

The quantum yield of NADH oxidation (Φox) was estimated from the oxidized NADH within 10 min (Figure ) and the photon fluence absorbed by dyes (FluAP, unit: nmol min–1; Supporting Information) as followswhere [NADHox] is the amount of oxidized NADH (unit: nmol). The calculated values are listed in Table . NMB demonstrated the highest activity in the phenothiazine dyes used. This result can be explained by the fact that reduced NMB is easily reoxidized and accelerates the redox cycle (described later).

Table 2

Quantum Yields of the NADH Oxidation Processes by Photoirradiated Phenothiazine Dyes through Electron Transfera

dyes	Φox	ΦET	Φrec
MB	3.0 × 10–2	2.7 × 10–4	1.1 × 102
AZA	5.4 × 10–2	3.4 × 10–4	1.6 × 102
AZB	5.3 × 10–2	2.5 × 10–4	2.1 × 102
NMB	1.7 × 10–1	3.4 × 10–4	5.0 × 102

Φox: the total quantum yield of NADH oxidation. ΦET: the quantum yield of the electron transfer from NADH to photoexcited phenothiazine dyes. Φrec: the quantum yield of further reaction to form NAD+. The sample solution containing 5 μM MB and 100 μM NADH with or without 10 mM NaN3 in a 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an LED (λmax= 659 nm, 0.5 mW cm–2). These quantum yields were calculated using eqs –9.

Mechanism of Photosensitized NADH Oxidation: Chain Reaction and Rate-Determining Step

The above-mentioned NADH oxidation can be explained by the electron transfer from NADH to the photoexcited state of phenothiazine dyes as shown in Figure . The collision between the S1 (or T1) states of dye molecules and NADH is the initial process of this electron-transfer reaction. In this section, the kinetics of NADH photooxidation are discussed under the assumption that the S1 state of phenothiazine dyes induces oxidative electron transfer. Because the fluorescence lifetime (τf) (the S1 state lifetimes) of these dyes (MB: 0.37 ns; AZA: 0.46 ns; AZB: 0.34 ns; and NMB: 0.46 ns) was barely affected by NADH, the efficiency of this electron-transfer reaction was too small to be determined under this experimental condition. Therefore, the possible quantum yield (ΦET; Table ) of this electron transfer is expressed as followswhere kdif is the diffusion control reaction limit (7.4 × 109 M–1 s–1) in this experimental condition, [NADH] is the concentration of NADH, and k0 (=1/τf) is the deactivation rate constant expressed using the τf values (same as the S1 state lifetime). This electron transfer produces NAD•, which undergoes further reaction to produce NAD+ (Figure ). Using the quantum yield of this further reaction (Φrec), the Φox can be expressed as followsSince the T1 state may contribute to NADH oxidation, the estimated Φrec values are the maximum limits. Although the actual Φrec values may be smaller than these listed values (Table ), the estimated values were much larger than 1, suggesting a chain reaction. A similar phenomenon was reported previously in the case of NADH oxidation photosensitized by porphyrin P(V) complexes.[24]

Figure 4

Relaxation processes for the photoexcited phenothiazine dyes and the photosensitized NADH oxidation.

The proposed mechanism of NADH photooxidation (photoredox cycle) is shown in Figure . Figure shows that the reduced forms of phenothiazine dyes are reoxidized to the cationic forms, resulting in the construction of a redox cycle. The reaction rate coefficients of the initial process (k1) and the reoxidation of reduced dyes (k2) are expressed as followsandwhere [Dye] is the concentration of the cationic form of the phenothiazine dye, [Dyered] is that of the reduced form, and [O2] is the dissolved oxygen concentration (260 μM under this experimental condition).[41] The time profile of the photosensitized NADH oxidation was analyzed using a numerical calculation to estimate these rate coefficients (Table ). The values of k2 are much smaller than those of k1, and a good relationship between k2 and Φox (correlation coefficient: 0.98) was observed (Supporting Information). These findings demonstrate that the reproduction process for cationic dyes is the rate-determining step in this photosensitized reaction. The highest photooxidative activity of NMB can be explained by the fast reoxidation of the reduced form of NMB. The calculation using density functional theory (DFT) showed that the ionization energy of reduced NMB (neutral radical form; 6.09 eV) is smaller than that of other dyes used in this study (MB: 6.10 eV; AZA: 6.20 eV; and AZB: 6.16 eV), supporting the fast reoxidation of the reduced NMB.

Figure 5

Proposed mechanism of NADH decomposition by phenothiazine dyes through photoredox cycle and chain reaction.

Table 3

Kinetic Parameters of NADH Oxidation Photosensitized by Phenothiazine Dyesa

dyes	k1 (M–1 s–1)	k2 (M–1 s–1)	k2′ (M–1 s–1)
MB	25.0	0.28	2.42
AZA	21.7	0.70	10.0
AZB	33.3	1.25	11.7
NMB	17.5	4.50	66.7

The sample solution contained 10 μM phenothiazine dyes and 100 μM NADH with or without 0.1 μM Cu2+ in a 10 mM sodium phosphate buffer (pH 7.6). The irradiation condition was the same as that in Table . The k2′ is the rate coefficient of reoxidation in the presence of 0.1 μM Cu2+. To analyze the NADH photooxidation by phenothiazine dyes with Cu2+, the same values were used for the k1 in this table.

Formation of Superoxide during the Photosensitized Reaction

The above-mentioned mechanism (Figure ) predicts the formation of O2•– in the presence of an oxygen molecule.[24,42] The formation of O2•– was evaluated using the cytochrome c reduction method (Supporting Information). The order of O2•– formation rates in this experimental condition was as follows: MB (9.6 × 10–2 μM s–1) > AZB (3.4 × 10–2 μM s–1) > NMB (3.0 × 10–2 μM s–1) > AZA (2.7 × 10–2 μM s–1). The kinetics of O2•– formation are complex because the possible other processes of O2•– formation are the reoxidation of reduced dyes and the oxidation of NAD•, and O2•– is consumed to produce hydrogen peroxide (H2O2).[29,35,36] Therefore, this order of O2•– formation rates could not be explained well; however, these results support the proposed mechanism in Figure and suggest the secondary formation of reactive oxygen species, O2•– and H2O2, during these photosensitized reactions. A similar result has been reported previously.[24]

Effect of a Copper Ion on the Photosensitized Reaction

The addition of a copper ion (Cu2+) markedly enhanced the photosensitized NADH oxidation by phenothiazine dyes (Figure ). Copper is an important endogenous metal.[43,44] It has been reported that Cu2+ can reoxidize the reduced form of MB to the initial cationic form.[29] In the presence of 5 μM Cu2+ (equimolar quantity of dyes in this experimental condition), the reduced phenothiazine dyes were immediately reoxidized to their cationic forms (data not shown), suggesting that this reaction is very fast. Analysis of the time profile using a method similar to that in Figure indicated the increase of k2 values by Cu2+ and enhancement of the reoxidation process of reduced dyes (Table ). The role of Cu2+ could be speculated as shown in Figure , similar to the literature.[29] The copper ion catalyzes the reoxidation of the reduced dyes. Because the reoxidation of the reduced dyes by Cu2+ is very fast, the reproduction of Cu2+ from Cu+ through the oxidation by oxygen is the rate-determining step. These results suggest that the endogenous metal ions play an important role in photosensitized NADH oxidation. In the case of NMB, the rate coefficient of reoxidation became larger than that of the electron-transfer rate coefficient.

Figure 6

Time profile of the NADH decomposed by the photosensitized reaction of phenothiazine dyes with Cu2+. The sample solution containing 5 μM phenothiazine dye (MB, AZA, AZB, or NMB), 100 μM NADH, and 0.1 μM Cu2+ in a 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an LED (λmax = 659 nm, 0.5 mW cm–2).

Figure 7

Scheme of the accelerated reoxidation process by Cu2+.

Conclusions

Phenothiazine dyes photosensitized NADH oxidation through electron transfer. The S1 (or T1) state of phenothiazine dyes extracts electrons from NADH through diffusion and collision. The reduced forms of these phenothiazine dyes undergo reoxidation to the original cationic forms, leading to the construction of a photoredox cycle. This reoxidation process is the rate-determining step in the photosensitized NADH oxidation. The NAD• formed through this electron transfer can trigger the chain reaction of the NADH oxidation (Figure ). Secondary reactive oxygen species, O2•– and H2O2, can be produced during this chain reaction in the presence of oxygen molecules. Copper ions enhance the photoredox cycle through reoxidation of the reduced forms of phenothiazine dyes. Endogenous metal ions may play an important role in photosensitized NADH oxidation in biological environments. In this study, NMB demonstrated the highest photooxidative activity due to the fast reoxidation process of the reduced form of NMB. Electron-transfer-mediated oxidation and the role of endogenous metal ions may be important in the photosterilization mechanism.

Experimental Section

Materials

MB and KI were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). AZA, AZB, and superoxide dismutase (SOD) were from Sigma-Aldrich Co. LLC. (St. Louis, MO). Copper chloride, cytochrome c, and NaN3 were from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). NMB was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). NADH and sodium phosphate buffer (0.1 M, pH 7.6) were from Nacalai Tesque, Inc. (Kyoto, Japan). These reagents were used as received.

Measurements

The absorption spectra of phenothiazine dyes and NADH were measured with a UV–vis spectrophotometer UV-1650PC (Shimadzu, Kyoto, Japan). The fluorescence spectra of the samples were measured with an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The τf of phenothiazine dyes was measured using a time-correlated single-photon counting method with the TemPro Fluorescence Lifetime System (HORIBA, Kyoto, Japan). Laser excitation at 637 nm was achieved using a diode laser (NanoLED-635L, HORIBA) at a repetition rate of 1.0 MHz. The experimental error of this measurement is within 0.01 ns.

Determination of Photosensitized NADH Oxidation

The sample solution containing phenothiazine dyes and NADH in a 10 mM sodium phosphate buffer (pH 7.6) was irradiated with a light-emitting diode (LED) (λmax= 659 nm, 0.5 mW cm–2, CCS Inc., Kyoto, Japan). The intensity of the LED light source (unit: mW cm–2) was measured with an 8230E optical power meter (ADC Corporation, Tokyo, Japan). The FluAP was estimated from the observed intensity of the LED and the absorption spectrum of the dyes (Supporting Information). The photosensitized NADH oxidation by phenothiazine dyes was evaluated by measuring the absorbance of NADH at 340 nm as previously reported.[24] The [NADHox] was estimated from this absorbance change (Supporting Information).

Calculations

The optimized structure and energy of reduced phenothiazine dyes (neutral radical forms) were calculated using the DFT method at the ωB97X-D/6-31G* level utilizing the Spartan 18′ (Wavefunction Inc., CA).

Measurement of Superoxide Formation

The quantity of superoxide (O2•–) formation during the photosensitized reaction was determined using the cytochrome c reduction method.[45] The sample solution containing 50 μM ferricytochrome c, 100 μM NADH, and 5 μM phenothiazine dyes with or without 150 U mL–1 SOD in 1.2 mL of 10 mM sodium phosphate buffer (pH 7.6) was irradiated. The absorption at 550 nm (molar absorption coefficient: 21 100 M–1 cm–1)[45] was measured with the UV–vis spectrophotometer UV-1650PC (Shimadzu), and the quantity of reduced cytochrome c was then calculated to determine the O2•– formation.