organic solution interface in the absence and presence of oxygen

ELSEVIER

Journal of ElectroanalyticalChemistry438 (1997) 147- 15!

Reduction of flavin mononucleotide at the aqueous[organic solution interface in the absence and presence of oxygen 1 Mitsuko

Suzuki

~, M a s a k a z u

Matsui

~, S o f i n K i h a r a b, °

a The Institute for Cheraical Research, Kyoto Unicersily, Ufi. Kvoto 611. Japan b Deportment of Chemistry, Kyoto Institute of Technology Afatsugasaki~ Sake'o, Kvoto 606, Japan

Received9 August 1996;receivedin revisedform I October 1996

Abstrnct The electron transfer at the ;aterface between an aqueous solution (W) containing flavin mononnc!eolide (FMN) and an organic solution (Org) containing decamethylferrocene in the absence and presence of oxygen was studied by polarography. The reduction of FMN in the absence of oxygen at the WlOrg interface differed from that observed by voltarnmea'y at metal or carbon electrodes. The reduction of FMN proceeds as a one-electron process at the WlOrg interface, whereas a two-electron process is observed at Lhe metal or carbon electrodes. In the presence of oxygen, the reduction of oxygen by chemical reaction with the reduced FMN was observed at the W~Org interface. Mechanisms of reduction processes of FMN and oxygen at die WJOrg interface arc discussed. © 1997 Elsevier Science S.A. Keywords: Flavin mononucleotide;Organic solution; Reduction;Oxygen

1. Introduction Flavin (riboflavin (RF), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD)) is the prosthetic group of the flavoenzymes, which are present in both the aqueous and membrane phases in cells and catalyze several one- or two-electron redox reactions [l]. The reduction of flavin has been studied widely by voltammetry at metal or glassy carbon (GC) electrodes in the absence of oxygen, and the reduction of RF, FMN and FAD has been reported to proceed as a two-elec~on process [2]. The two-electron reduction wave of FAD in the presence of oxygen was somewhat larger than the sum of the limiting current for the reduction of FAD in the absence of oxygen and that for oxygen. This result was attributed to the catalytic reduction of oxygen by redvced FADH 2 [3]. Much accelerated catalytic reduction of oxygen was observed when FAD is immobilized as a multilayer film instead of a plane GC electrode [3]. The reac-

tions of reduced RF, FAD or FMN with oxygen (~O z) in aqueous solutions were investigated widely by pulse radiolysis and flash phot~lysis, and reaction processes have been proposed to involve flavin radicals and 0 2 - , or a complex of reduced fiavin with oxygen [4-8]. In the present work, the reduction of FMN in the aqueous solution (W) at the interlace between W and 1,2-dichloroethane (DCE) containing a reducing agent has been investigated in the absence and presence of oxygen, and the ~duction processes of FMN and oxygen are discussed. In this connection, the aqueous (W~.~rganic (Org) interface has often been regarded as a convenient model of the aqueous[membrane interface [9]. Several electron transfer reactions at the W~3rg interface have been investigated voltammetdcally [10-13], with subsequent theoretical an~ysis of the voltammogram and of the energy transformation at the interface [14-17].

2. Experimental

• Correspondingauthor. i This paper was presented at the International Symposiumon Electron Transfer in Protein and SupramoleculatAssemblies at Interfaces held in Shonan Village, Kanagawa, Japan on 17 to 20 March 199~, 0022-0728/97/$17.00 @ 1997 ElsevierScience S.A. All fights reserved. i'll S0022-0728(96)04983-2

2.1. P o l a r o g r a p h i c m e a s u r e m e n t s

The polarograms at the W[DCE interface were recorded by current-scan polarography at the aqueous electrolyte

148

M. Suzuki et al. / Journal of Electroanalytical Chemistry438 (1997) 147-151

solution dropping electrode using instruments identical with those described previously [12,18]. The phase W containing FMN, pH buffering agents and supporting electrolyte was forced upwards dropwise into DCE containing a reducing agent and the supporting electrolyte through a glass capillary [12]. Prior to the polarographic measurement, both W and DCE were saturated with nitrogen, air or oxygen by bubbling these gases. The polarogram was recorded by scanning the current at a rate of 0.5 g A s - J and dropping W at a flow rate of 0.020 ml s - J and a drop time of 4.2 s at open circuit, unless noted otherwise. The supporting electrolytes in W and DCE were 0.5 mol dm -3 Na2SO 4 and 0 . 0 4 m o i d m -3 tetraheptylammonium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (THepA +. T B C F 3 P h B - ) respectively. The pH of W was adjusted with 0.04 mol d m - 3 phosphate buffer. The potential difference A E at the WlOrg interface was measured with "~heaid of an AgIAgC! electrode in W and a tetrapi,enylborate ion-selective electrode in DCE respectively [18]. In the lbllow~ng, A E thus measured is converted to the TPhE scale based on the tetraphenylarsonium-tetraphenylborate assumpt,.'.m. On this scale, 0 V vs. TPhE corresponds to the potential where the transfer energy is zero [18]. Polarographic measurements were carried out at 25 ± 0.5°C.

2.2. Chemicals T H e p A + - T B C F 3 P h B - and tetraheptylammonium tetraphenylborate were synthesized and purified according to the procedure described previously [18]. The sodium salt of FMN (Tokyo Kasei, guaranteed grade, lot no. AX01) and bis(l,2,3,4,5-pen'tamethylcyclopentadienyl)iron (Aldrich, lot no. 37, 854-2), commonly called decamethylferrocene and abbreviated as DMFC hereinafter, was used as received. All other chemicals were of reagent grade quality.

3. Results

In the following, we define the transfer of such negatively charged species as an electron or an anion from W to DCE and that from DCE to W to be the cathodic and anodic reactions respectively.

3.1. Polarograms for electron transfer from D M F C in DCE to F M N in W in the absence of oxygen The pola_rogra_m shown as curve 1 in Fig. 1 was recorded at the interface between W containing 10 -4 m o l d m -3 FMN (pH 7.1) and DCE containing 0.02 m o l d m -3 DMFC after deaeration of both W and DCE solutions. The anodic wave is attributable to the electron transfer from DMFC in

2

I I0 l.tA

~ i"

~

-0.2

0.2 E/V vs. TPhE

0

Fig. I. Current-scanpolarograms for the electron transfer from DMFC in DCE to FMN in W observed with the deaerated (curve I), air-saturated (curve 2), and oxygen-saturated (curve 3) solutions. 10-4 moldm -3 FMN+0.032moldm -3 Na2HPO4 +0.008moldm -3 NaH2PO4 + 0.5moldm -3 Na2SO4 in W, 0.02moldm -3 DMFC+0.04moldm -'~ THepA+.TBCF3PhB- in DCE. --- Background current in the absence of DMFC or FMN.

DCE to FMN in W, because (1) no anodic waves were observed in the absence of either FMN in W or DMFC in DCE, (2) FMN and its reduced form are highly hydrophilic and hence are hardly expected to transfer from W to DCE, and (3) the cathodic wave was not observed in the polarogram when DCE contained the oxidation product of DMFC, i.e. decamethyiferricenium cation, and W contained only the supporting electrolyte and pH buffer. The values of the anodic limiting currents measured under deaerated conditions are summarized in Table I. When the concentration of DMFC in DCE was higher than 0.01 m o l d m -3, the limiting current was almost independent of the concentration of DMFC at an 3, pH of W. Hence, polarograms were measured using 0.02 m o l d m -3 DMFC unless mentioned otherwise. The limiting anodic current / r at pH > 8 was almost independent of pH and proportional to both the square root

Table 1 Half-wave potentials AEI/2 and limiting currents I t of polarographic waves of the clectron transfer between DMFC in DCE and FMN in W under deaerated conditions; for other conditions see the caption to Fig. 2 pH A El~.2/V vs. TPhE 11/p.A 3.22 3.84 4.56 5.28 5.96 5.92 6.6 7.13 7.37 7.56 8.03 8.44 8.5 8.62

-0.115 "- 0.08 - 0.03 0.005 0.045 0.03 0.07 0.1 0.12 0.13 0.15 0.145 0.15 0.15

11 I1

I 1.5 10.5 10.5 10.5 11.5 12 10 t1 8.5 8.5 8 8

M. Suzuld et al. / Journal of ElectroanalyticalChemistry 438 (1997) 147-151

0.1 0 -0.1 4

6 pH

8

Fig. 2. Effect of pH on the half-wave potentials AEt/2 of polarographic waves for the electron transfer from DMFC in DCE to FMN in W observed with the deaerated (curve 1) and air-~aturated (curve 2) solutions. 10-4moldm -3 FMN+O.O4moldm-3 pH buffer agents+ 0.5moldm -3 Na2SO4 in W, 0.02moldm--~ DMFC+0.04raoldm -3 THepA+.TBCF3PhB- in DCE.

of the height of the aqueous solution reservoir and the concentration of FMN in W in the range from 5 × t0 -5 to lO -3 moldm -3. Therefore, the transfer of electrons from DMFC in DCE to FMN in W (pH > 8) was considered to be controlled by the diffusion of FMN in W. A comparison with the diffusion-controlled limiting current at pH > 8 (8.2 p.A for 10 -4 moldm -a FMN) and calculated from the diffusion coefficient of FMN in W observed at the dropping mercury, electrode ( D = 6.3 X 10 -6 cm 2 s - i ) [19] and Ilkovi~'s equation indicates that the number of electrgns involved in tl',e r:duction process at the W[DCE interface is n -- 1. The analyses of polarographic waves at pH > 8 based on eq. (14) of Ref. [12] or ¢q. (29) of Ref. [14] support this result (n = l). The limiting currents at pH < 8 are larger than those at pH > 8 at a given concentration of FMN, as seen in Table I. The limiting currents at pH < 8 were independent of pH, and proportional to the concentration of FMN in the range from 3 X 10 -5 to 10 -3 moldm -3. The values of half-wave potentials A.~'t/2 obtained under deaerated conditions are summarized in Table 1. The half-wave potentials of polarograms under deaerated conditions shifted by 56 m V / p H unit to more. negative potentials with decreasing pH in the range 3 < pH < 8 and almost independent of pH in the range 8 < pH, as shown in Fig. 2. The pH dependence indicates that the electron transfer reaction at the WE}CE interface at pH < 8 is followed by the protonation of reduced FMN in W and that the number of protons added is equal to the number of electrons transferred (n = 1). An inflection point appeared at around pH 8, being near to the p K 8.3 of FMNH" (where K is the dissc~:.iation constant) [6], but the inflection point related to FMNH 2 (pK t 6.7) [6] was not observed. Taking into account the above mentioned facts, the reduced species of FMN are deduced to be FMN - at pH > 8 and FMNH" at pH < 8, rather than FMNH 2 which

I*~9

was reported as the reduction product at the metal or GC electrodes. Although ferrucene dimethylferrocene ~ c l t~rathiafub valene were examined as reducing agents in DCE imtead of DMFC, no distinct l~larographic waves due to the electron transfer between these reagents in DCE and FMN in W were observed at any pH between 4 and 9. The reduction of FMN at the W ~ t 2 E interface is differem from that at metal electrodes. It has been reported [2] that FMN shows a two-electron reduction and ox/dation waves at metal electrodes, such as the dropping ng~rcury and Pt electrodes, and that the inflection points in the half-wave l;atential-pH relation can be found at pH about 6.8 and 9.65. Besides, a prewave and a postwavc were observed [2]. The difference between reduction of FMN at the metal electrodes and the WIDCE interface can be linked to abe adsorption of FMN or its reduced forms to the electrodes [2].

3.2. Polarograms f o r electron transfer f r o m D M F C in DCE to FAIN in W in the presence o f oxygen

The polarogram represented by curve 2 in Fig. 1 was recorded at the interface between air-satiated W of pH 7.1 containing 10 -4 moldm -3 FMN and air-saawated DCE containing 0.02mnldm -3 DMFC, The polarogram shown as curve 3 w~s measured under the same conditions as that for curve 2, but using W and DCE saturated with oxygen instead of air. Polarographic waves of 10 -4 moleh'n -3 FMN at pH > 8 and 0.02moldm -3 DMFC in DCE measmed under air- and oxygen-saturated conditions merged with the final rise of the background current. Here, the final rise means a large positive current limiting the potential window. When DCE contained more than 0.01 moldm -3 DI~IFC, the limiting currents of the polarographic waves using the air- or oxygen-~aturated solutions were almost independent of the concentratio,n of DMFC at any pH of W. Hence, polarograms were mea.~med using O.02moldm -3 DMFC in the following. When the pH was between 6.5 and 8, the I I in polarograms under air-saturated conditions was about seven times la,,ger than that with deaerated solutions, or the lj under oxygen-saturated conditions was eleven times larger than that with deaerated solutions, as shown in Fig. 1. The limiting current was propo~onal to ~ concentration of ~MN in the range from I0 -s to 5 × 10 -4 tool dm -3, but almost iadependent of the height of the aqueous solution reservoir The slopes of log 1 vs. log t ( / = c u r r e n t , t = time during growth of the individual aqueous solution drop) curves at a potential in the limiting current region weTe about 0.5 in the pH range between 5 and 8. These characteristics suggest that the limiting current observed under air- or oxygen-saturated conditions is a catalytic current due to the regeneration of FMN in the vicinity of the W ~ C E interface. Since the catalyac current was not

150

M, Suzuki et al. /Journal of Electroanalytical Chemistry 438 (1997) 147-151

observed in the absence of oxygen, it can be concluded that FMN is reproduced through the re-oxidation of reduced FMN by oxygen. In this connection, the polarographic wave due to the direct electron transfer from DMFC in DCE to oxygen in W (pH 3 to 8) in the absence of FMN was not observed in the present work, although Cunnane et al. [20] reported the electron transfer across the interface between W containing oxygen and sodium citrate and DCE containing DMFC and tetraphenylarsonium tetrakis( p-chlorophenyl)borate. The magnitude of the limiting current 1] under airsaturated conditions was the same as that under deaerated conditions at pH < 4, but it increased with increasing pH at pH > 4, and was almost independent of pH in the range pH > 6.5, as illustrated in Fig. 3. This indicates that the oxidation of reduced FMN by oxygen takes place at pH > 4, and that the oxidation reaction is speeded up at higher pH. In the pH range between 3 and 4.5, A E t ! ~- of polarograms under air-saturated conditions shifted by 57 m V / p H unit to more negative potentials with decreasing pH, in agreement with those under deaerated conditions, as shown in Fig. 2. At pH higher than 4.5, the half-wave potentials in air-saturated solutions were more positive than those with deaerated solt~tions. The half-wave potentials shifted by 65 m V / p H unit to more negative potentials with decreasing pH when pH > 5.5, suggesting that the electron transfer at the WIDCE interface and the following protonation in the presence of oxygen proceed similarly as in the absence of oxygen. Table 2 summarizes the values of A El~2 and 11 obtained with air-saturated solutions. When both W and DCE solutions were saturated with oxygen instead of air. 11 at pH 7 is only about 1.5 times higher than that . ose,ved with the air-saturated solutions, as seen in Fig. 1, though the oxygen concentration in W is about five times higher than that in the air-saturated solution3 [21].

80 60 •

• Qeee~ o

3

-0.115 -0.09 - 0.07 - 0.06 - 0.03 - 0.025 - 0.005 0.035 0.035 0.07

11.5 13 12 14.5 17 23.5 25 38 39 55

(I.092

64

0.11 0.115 0.125 O.14 0.16 0.16 0.175 0.185 0.20 0.21

67 72 81 78 83 86 78 87 83 81

When H 2 0 2 was added to W at a concentration of 0.08 or 0 . 8 m o l d m -3 under nitrogen atmosphere, the polarograms were identical with those observed with deaerated solutions, and the catalytic current was not observed. This indicates that H 202, the reduction product of oxygen, does not oxidize F M N H ' / F M N ' - to FMN at the WIDCE interface, and F M N H ' / F M N ' - does not reduce H202.

4. D i s c u s s i o n

F M N ( W ) + D M F C ( D C E ) ~- F M N - ( W )

0

20

3.1 3.53 3.83 4.05 4.43 4.51 4.58 5.02 5.23 5.65 5.89 6.09 6.41 6.43 6.77 6.89 7.01 7.13 '•.35 7.45 7.79

In the present work, the reduction of FMN in W with DMFC in DCE at the W IDCE interface is demonstrated to proceed as a one-electron process. Based on this result, and recalling that the oxidation of DMFC in an organic solvent produces a stab!e cation radical, DMFC '+ [22,23], a conclusion can be drawn that the anodic wave of curve l in Fig. 1 observed in the absence of oxygen is caused by the electron transfer from DCE to W:

Q I~I~:Q0

<

Table 2 Half-wave potentials AEI/2 and limiting currents II of polarographic waves of the electron transfer between DMFC in DCE and FMN in W under air-saturatedconditions; for other conditions see the caption to Fig. 2 pH A E~/:/V vs. TPhE II/p,A

. , 5

pH

I

et~.~ 7

+ DMFC'+ ( D C E ) 9

Fig. 3. Effect of pH on the limiting currents 11 of polarographicwaves for the electron transfer from DMFC in DCE to FMN in W observed with the deaerated (curve 1) and air-~turated (curve 2) solutions. Conditions: see caption to Fig. 2.

(l)

where (W) and (DCE) denote species in the W and DCE phase respectively. The elec~on transfer may be followed by protonation of F M N ' - in W at pH < 8 ( p K of F M N H ' 8.3): F M N ' - + H ÷ ~ FMNH"

(2)

M. Suzuki et al. / Journal of Electroanalytical Chemistry 438 (1997) 147- ! 5 i

At pH < 8, I t larger than at pH > 8 was observed, which can be explained by assuming that the regeneration of FMN occurs through the disproportionadon of FMNH ": 2 F M N H ' ~ FMN + FMNH 2

(3)

The catalytic current appeared at pH > 4 under airsaturated conditions. The primary pro~:ct of the reduction of oxygen in the phase W at pH > 4 is assumed to be O 2or H e 2 in the present work, referring to works in which 0 2- was reported as the intermediate in the reduction of oxygen to H 2 0 ? by the reduced FMN [4-6]. Taking into account that p K of H e 2 due to Eq. (4) is 4.45 [24,25] and the catalytic current appears at pH > 4, the reduction of oxygen by FMNH" in W at pH > 4 may be expressed by Eq. (5): HO~ ~ H + + 0 2 -

(4)

FMNH + 0 2 ---,FMN + O2- + H +

(5)

Here, the reaction FIvlNH + 0 2 ~ FMN + HO.~

(6)

is considered fairly slow, because the catalytic reaction is not observed at pH < 4. In this connection, the p K of the species which take part in the catalytic reaction, such as oxidized, radical and reduced forms of FMN, do not exist around pH 4. When the pH was between 6.5 and 8, I t under airsaturated conditions did not increase with increasing pH, and was almost independent of pH. In this pH range, the product of the disproportionation reaction of Eq. (3), FMNH 2, dissociates to F M N H - , because its p K is 6.7 [6], which accelerates the disproportionation reaction and may therefore lower the concentration of FMNH'. Consequently, the proportion of the catalytic reaction of F M N H with 0 2 [Eq. (5)] to the disproportionation reaction [Eq. (3)] may decrease compared with that with W of pH < 6.5. The intermediate product of the catalytic reduction of 0 2 , 0 2 - , can produce H202 through a chemical reaction with F M N H ' [cf. Eq. (7)], F M N ' - [cf. Eq. (8)] or H 2 0 : F M N H ' + 0 2- + H + ~ FMN + H 2 0 2

(7)

F M N ' - + 0 2 - + 2H +-* FMN + H202

(8)

5. Conclusion The electron transfer reaction from a reducing agent in Org to FMN in W was studied at the W[Org interface in

151

the absence and presence of oxygen. Although the reduction of FMN at metal or carbon electrodes in the absence of oxygen proceeds as a two-electron process to yield FMNH 2, the reduction at the W[DCE imerface is a oneelectron process yielding F M N H . In th~ presence of oxygen, the reduction of FMN gave a limiting current much larger than that in the absence of oxygen. This phenomenon was explained by considering the regenermion of FMN by the chemical reaction between F M N H and oxygen.

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