Electron transfer between viologen derivatives and the flavoprotein ferredoxin-NADP+ reductase

ELSEVIER

Bioelectrochemistry and Bioenergetics 38 (1995) 179-184

Electron transfer between viologen derivatives and the flavoprotein ferredoxin-NADP ÷ reductase M. Teresa Bes 1, Antonio L. de Lacey 2, Victor M. Fernandez 2, Carlos Gomez-Moreno 1,, 1 Departamento de Bioquiraica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain 2 lnstituto de Catalisis y Petroleoquimica, CSIC, Madrid, Spain Received 9 November 1994; revised 3 February 1995

Abstract The mechanism of electron transfer of several diquatemary salts of 4,4'-bipyridine and 2,2'-bipyridine with the FAD which is the prosthetic group of the protein ferredoxin-NADP + reductase from the cianobacterium Anabaena PCC 7119 was studied. Two different reactions involving the same compounds were studied: (i) the reduction of NADP ÷ by the enzyme using reduced viologens; (ii) reduction of the viologens using NADPH and the enzyme. Different catalytic rates were obtained in each case, with values very similar to the turnover numbers reported for other enzymatic reactions described for this enzyme. In all cases a correlation between the second-order rate constant and the difference in redox potential of the groups exchanging electrons was obtained in agreement with the outer-sphere electron transfer theory. Those cases in which anomalous behavior was observed corresponded to viologens with an electrical charge quite different from the rest. The reaction between a highly charged viologen and the enzyme was much more dependent on the ionic strength of the medium than is the case for those having lower or no charge, which is an indication of the modulating effect of the electrical nature of the reactant on the electron transfer process. Keywords: Ferredoxin-NADP + reductase; Viologens; Electron transfer

1. Introduction Electron transfer reactions are essential for biological energy transformation processes and usually involve the exchange of either one or two electrons between the prosthetic groups of proteins forming a functional transient complex. Despite the long distance that the electrons have to cover, these reactions are remarkably fast and highly specific with respect to the proteins involved. It is generally assumed that proteins exchanging electrons form a rather tight complex in which several amino acid residues of the protein interface are involved. It is also suggested that other amino acid residues could play a role in promoting the passage of the electron(s) between the redox groups in what can be considered a very interesting exam-

* Corresponding author. Elsevier Science S.A. SSDI 0 3 0 2 -45 9 8 ( 9 5 ) 0 1 8 1 7 - 4

ple of biological specificity through a mechanism of molecular recognition [1,2]. The high specificity of this phenomenon constitutes a drawback when the redox protein is required to act in an artificial environment. This is usually the case for an enzymatic assay in which a non-physiological substrate is used or when the interaction of a redox enzyme with an electrode is required. In these latter two cases a carrier molecule is required to mediate the exchange of electrons between the rather inaccessible prosthetic group in the enzyme and the substrate or the electrode. There are several recent reports in which structure-function relationships for non-physiological substrates of enzymes have been studied [3-5]. Viologens are traditionally considered as one of the most efficient molecules for this purpose [6]. A m o n g this group, methyl and benzyl viologen are the most commonly used in biochemical reactions [7-9]. The viologens exist in three main oxidation states, namely V 2÷, V ÷" and V ° (accepted nomenclature for methyl

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M.T. Beset al. / Bioelectrocheraistry and Bioenergetics38 (1995) 179-184

viologen). The first reduction step is highly reversible and the viologen undergoes a very obvious color change. Further reduction to the fully reduced state is less reversible, not only because the latter is frequently an insoluble species but also because it is uncharged. The negative redox potentials of viologens make them suitable for those redox reactions taking place in the range where dehydrogenase enzymes participate. Their ability, in the reduced state, to exchange one electron with molecular oxygen, producing the highly reactive superoxide radical, is applied commercially in the potent herbicide referred to as paraquat [10]. The importance of the reactions in which viologens participate makes it of interest to study the parameters that determine the efficiency of their participation as mediators in enzyme reactions. Such information would allow the design of the most efficient molecules for this purpose. Moreover, from a basic point of view, the understanding of the factors that determine the redox reaction rate constants as well as the molecular basis for their biological specificty/leads to further interest in this type of study. The most commonly used theory to study electron transfer reactions is that developed by Marcus and co-workers [11,12] which has been applied by Tollin et al. [13] to biological systems with the following conclusions: (1) reaction rate constants can be correlated with redox potential differences for at least several electron-exchanging proteins; (2) rate constants are strongly influenced by other parameters such as the exposure of the prosthetic group at the protein surface, the steric accessibility of the prosthetic group and electrostatic interactions involving charged groups principally at or near the reaction site. The flavoprotein ferredoxin-NADP + reductase (FNR) (EC 1.18.1.2) isolated from the cyanobacterium Anabaena PCC 7119 transfers electrons from reduced ferredoxin to NADP + on the reductive side of the photosynthetic reaction [14]. The NADP ÷ reduction reaction can be carried out in vitro by irradiating a preparation of broken chloroplasts with light [15]. FNR can also be assayed using NADPH as the electron donor to reduce either an artificial electron acceptor such as DCPIP [16] in a diaphorase activity or to ferredoxin and then to cytochrome c [17]. FAD, which is non-covalently bound to the protein, has a midpoint redox potential of - 3 7 6 m V / S H E at pH 8 [18,19]. In the present work we have used several viologen derivatives with different redox potentials and different electrical charges to study the rate of electron exchange with the enzyme FNR. Moreover, the reversibility of these reactions has allowed us to check these parameters in reactions in wh!,ch the reduced viologens act as electron donors to the enzyme as well as in those cases where these compounds act as electron acceptors of the enzyme. The results indicate that the main parameter controlling the rate of electron transfer is the difference in redox potential between the electron donor and acceptor. However, the reaction rates are also modulated by the number and nature of the charge(s) present in the molecule, probably because

of the electrostatic environment of the prosthetic group in the protein.

2. Materials and methods 2.1. Materials Anabaena PCC 7119 FNR was purified to homogeneity, as previously described [20], from cells that were grown autotrophically on nitrate. Its concentration was determined spectrophotometrically using an extinction coefficient of 9.4 mM -1 cm -1 at 459 nm. 1,1'-Dibenzyl4,4'-bipyridylium dichloride (BV) (Sigma), viologen propyl sulphonate (PVS) (Sigma), 1,1'-dimethyl-4,4'-bipyridylium dichloride (MV) (Serva) and 1,1'-trimethylene-2,2'-pyridylium dibromide (PDQ) (ICI) were used as received. Diethyl carbonic acid viologen bromide (DPV) and N,N'-di(g-aminopropyl)dipyridinium bromide (DAPV) were a gift from Dr. Cees van Dijk. 1-Propyl-l'-propionyl4-4'-dipyridyl dibromide (PPV) and 1,1'-dipropyl-4,4'-dipyridyl diiodide (PV) were synthesized as described in [21]. N-methyl-N'-(carboxypropyl)-4,4'-bipyridinium (APMV) was synthesized as described in Ref. [22]. All other chemicals were commercially available and of reagent grade. The redox potentials of the viologens used in the present work were determined by cyclic voltametry of 1 mM solutions in 50 mM 'Iris + HCI (pH 8) plus 100 mM KCI buffer as the supporting electrolyte under nitrogen in a conventional three-electrode electrochemical cell at 20°C. Glassy carbon (Le Carbon Lorraine, France) was used as a working electrode and Ag IAgCII 3M KCI as a reference electrode. The observed redox potentials quoted in Table 1 are not significantly different from those in the literature [9,21,23-28]. 2.2. Photoreduction o f N A D P +^

Steady-state experiments were carded out in a 1 ml bull-necked anaerobic spectrophotometer cell equipped with a side-arm. Anaerobic conditions were established by repeated cycles of evacuation and flushing with argon, which had been purified by passage over a heated BASF catalyst. Illumination (10000 lux) was performed using a 50 W halogen lamp fitted with an Oriel Corp. blue filter no. 59855, with a cut-off band between 320 and 550 nm. The decrease of absorbance in the blue spectral region of the one-electron reduced form of the viologens (Amax for each viologen in Table 1) as a consequence of NADP + reduction by FNR was followed using a thermostated Kontron Uvikon 860 spectrophotometer at 25°C. Reaction mixtures contained, in a final volume of 1 ml, 30 p~M proflavin, 0.2 mM oxidized viologen, 20 nM FNR, 20 mM EDTA and 1.15 mM NADP + in Tris + H O buffer (pH 8). Reduction of the viologens was performed prior to the addition of NADP + and FNR to avoid direct reduction of

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M.T. Bes et al. /Bioelectrochemistry and Bioenergetics 38 (1995) 179-184

Table 1 Mediator

(A) APMV (B) PVS (C) BV (D) DAPV (E) DPV (F) MV (G) PPV (H) PV (I) PDQ

R1

R2

El~ 2 a/mV

R3

H3C- O3S-(CH2) 3-

-(CH2)3-NH ~"

C6Hs-CH 2+ H3N-(CH2) 3-

-(CH2)-C6H 5 -(CH 2)3-NI--I~"

-430 - 410 -350 -385 -420 -460 --445

-(CH23-SO 3

- O O C - ( C H 2 ) 2-

-(CH2)2-COO-

H3C-

-CH 3

CHa-(CH2) 2CH3-(CH2) 2 -

-(CH2)2-COO-(CH2)E-CH 3

-455

-(CH2) 3-

R

-560

Viologen net charge

Spectral characteristics of radical

Oxidized

Radical

A/nm

e/mM- 1 cm - 1

3+ 0 2+ 4+ 0 2+ 1+ 2+ 2+

2+ 11+ 3+ 11+ 0 1+ 1+

602 602 600 602 600 600 604 604 493

13.6 b 12.8 [24] 7.5 [25] 14.3 [29] 8 [29] 14 [27] 12.6 [26] 12.7 [26] 3.2 [28]

Q y3+

R3 a Potential values are given versus the standard hydrogen electrode (SHE) at pH 8.0 and were determined as indicated in Section 2. b This work. *

N A D P + by proflavin. Their concentrations were determined using the extinction coefficients given in Table 1.

3. Results and discussion

Several viologen derivatives with different mid-point redox potentials (Table 1) were used as electron donors for the N A D P ÷ reduction reaction (Fig. l(a)). For this purpose the viologen derivative was photochemically reduced under anaerobic conditions in the presence of proflavin and EDTA. The concentration o f the reduced viologen was calculated from its absorbance in the region of 600 nm and then the reaction was started by mixing it with the enzyme and the oxidized pyridine nucleotide. The rate of the reaction was followed by the absorption change in the region of 600 nm, which is an indication of the rate of

2.3. Oxidation o f N A D P H

FNR activity was assayed using viologens as electron acceptors under aerobic conditions. The decay in absorbance at 340 nm was measured using a thermostated Kontron Uvikon 860 spectrophotometer at 25°C. The reaction mixtures contained, in a final volume o f 1 ml, 0.25 m M N A D P H , different amounts o f oxidized viologen (between 0 and 7 m M ) and 0.35 IxM F N R in 50 m M Tris + HC1 buffer (pH 8).

a) f , hv

EDTAox~ EDTAred-~

Pr° ~ p r O f o x ~ v i o l o g e n r ~ ~

pmflavin~ ~

-

~

~-viologenox~---~

~ ~-~

NADP ~

÷

NADPH

b) NADP+ ~ v i o l o g e n ~ NADPH~J

"-I

~

-~'-

viologenox-~.--." ~

O2 ~ 02-

Fig. 1. (a) Regeneration of NADPH by photochemically reduced viologens using FNR as the catalyst; (b) regeneration of NADP+ catalyzed by FNR in the presence of oxidized viologens.

182

M.T. Beset aL /Bioelectrochemistry and Bioenergetics 38 (1995) 179-184

oxidation of the viologen. Exclusion of the enzyme FNR eliminated the formation of NADPH. Fig. 2 shows the dependence of the rates of viologen oxidation on substrate concentration for the FNR-mediated reaction. Different rates of NADP + reduction were obtained for each viologen and also by changing the concentration of each viologen. This is an indication that the transfer of electrons between the viologen radical and FNR is the rate-limiting step in this reaction. The highest value measured (4100 min -1) corresponded to methyl viologen. This rate is of the same order of magnitude as the turnover number reported for the NADPH-cytochrome c reductase and for the diaphorase activity assayed with dichlorophenol-indophenol, which have been described as typical assays for this enzyme [18]. The high rates obtained for the reaction between reduced viologens and FNR are indicative of the efficient interaction between the two species, since the reactions involving the viologens were measured at non-saturated viologen concentrations because limitations in the experimental procedure prevented the use of very high concentrations of the reduced substrate. In each case second-order rate constants were determined from the plots of the reaction rates versus the viologen concentration. A semilogarithmic plot of these second-order rate constants versus the difference between the mid-point redox potentials of the corresponding viologen and the NADP + / N A D P H couple ( - 0 . 3 5 0 V / S H E at pH 8.0) shows a general trend indicating an increase in the rates of electron transfer with increasing difference between the redox potentials of the viologen acting as electron donor and the cofactor that becomes reduced (Fig. 3). The Marcus theory of electron transfer [12] has been successfully used by Tollin et al. [13] to correlate rate constants for the reduction of electron transfer proteins by reduced flavins with redox potential differences and by Kulys and co-workers for the oxidation of flavoproteins with quinones [3] and other redox mediators [5]. Fig. 3

4100 3280 2460 1640 ~o 820

E I--

0

0.000

•

.

0,055

!

0,110

.

g

0o165

.

0°220

[viol.]red./mM Fig. 2. Rates of NADP + reduction catalyzed by FNR with different viologcns photochemica]ly reduced as electron donors: • MV; r-1 PV; "Jr PPV; [] PVS; • DPV; zx APMV, [ ] BV; • DAPV.

10

7

10 6 H~ F

¢N

105

D 104 0

I

i

I

100

200

300

400

hEm8/mV

Fig. 3. Semilogarithmicplot of the apparent second-orderrate constants for electron transfer from free viologen semiquinones to NADP÷ in the presence of FNR versus the difference between the redox potentials (at pH 8.0) of the NADP+/NADPH couple and the viologens.The letters on the curves are defined in Table 1. The solid curve line is the plot of the semi-empirical Marcus equation with VET=l.6x108 M-l and AG#= 4.9 kcal tool-1.

shows the fit of our experimental data to the empirical equation given by Marcus [30]. In k2 = In Vex +

nFAE° - ( AG~o)/In 2) In{ 1 + exp[ nF ln(2)AE°/AG~o)]} RT

(1) The solid curve in Fig. 3 is the theoretical curve based on this equation that best fits our experimental data. The values obtained for VET (1.6 × 10 a M - i ) and AG # (4.9 kcal mol-1) are very similar to those obtained by Tollin et al. [13] for electron transfer proteins with different prosthetic groups. It is apparent from Fig. 3 that points H and F lie above the theoretical curve, while points A and D lie below it. The higher activity displayed by viologens H and F could be related to their aliphatic lateral chains which would require less solvent rearrangement during the electron transfer step than other viologens with charged groups. Points A and D correspond to viologens with amine groups in their lateral chains. These amine groups are protonated at pH 8.0 and consequently they carry more positive charges in the radical form. Electrostatic repulsions could decrease the equilibrium constant for the complex formation and therefore the value of the frequency factor V E T , which is a measure of the intrinsic probability that the redox reaction will occur [13]. The determination of the rate of the reaction in which the viologen acts as electron acceptor is more difficult since all the viologens used have more negative mid-point 'redox potentials than the pyridine nucleotide used as substrate and the FAD group in the enzyme. For this purpose the reaction was displaced towards the viologen reduction

M.T. Beset aL / Bioelectrochemistry and Bioenergetics 38 (1995) 179-184

1250' E ¢O ..Q

1000'

750'

E r- 500' C_ 0

>

0 IC: f,.-

I--

250' 0 0.00

0.55

1 .10

1.65

2.20

[viol.]ox./mM Fig. 4. Rates of NADPH oxidation catalyzed by FNR with different viologens as electron acceptors: • PDQ; [ ] DPV; • PPV; • PV; r, MV; [] APMV; ,A- DAPV; [3 BV.

by using molecular oxygen as the final electron acceptor (Fig. l(b)). In the absence of viologen, electron transfer between FNR and oxygen was completely absent. Fig. 4 shows the dependence of the rates of viologen reduction on substrate concentration for the FNR-dependent reaction. Lower rates were obtained for NADPH oxidation in this situation, with the highest value corresponding to benzyl viologen which has the most positive redox potential. Again, the rates increased proportionally to the concentration of the viologen present, indicating that the interaction between FNR and the oxidized viologen was the limiting factor. The second-order rate constants for these reactions are plotted in Fig. 5 versus the difference between the mid-point redox potentials of the viologen and the NADP+/NADPH couple. Note that, since the differ10e

ences have negative values, they are plotted in the opposite direction. The points in this plot could also be fitted to the Marcus equation with the parameters VET = 3.2 × 10 s and AG # = 4.4 kcal mol- 1. However, one point (E) is far from the line. This corresponds to DPV which, as Table 1 indicates, is the only compound assayed as an electron acceptor for this reaction, showing no positive charge in the oxidized state. The above results indicate that the main factor controlling the rate of electron transfer between the FAD group in FNR and an external viologen with which it exchanges electrons is the difference between the mid-point redox potentials. It is possible that other factors, such as the electrostatic interaction between the carrier molecule and the redox group in the protein, may modulate this main effect. In order to evaluate this further, three viologen molecules with different charges, namely APMV (3 + ), MV (2 + ) and DPV (zero), were used as electron acceptors in this second reaction and the effect of the ionic strength of the medium on the rate of electron transfer was studied. Fig. 6 shows that, whereas increasing the ionic strength appreciably reduces the rate of electron transfer for the highly charged molecule APMV, ionic strength has almost no effect for the uncharged DPV and an intermediate effect for MV. On the basis of the above results we can conclude that soluble viologen compounds with different electrical charges and redox potentials exchange electrons with FNR from Anabaena PCC 7119 with an efficiency that strongly depends on the difference between the mid-point potentials of the redox groups involved. This effect is modulated by other physicochemical characteristic of the molecule, such as the charge and the hydrophobicity, which can become rather important, producing a very strong deviation of the behavior expected from the thermodynamic characteristics. Furthermore, despite the non-physiological nature of the

105

T T

~E

1600 , ~ . . ~ \

104

183

....-..

120o

,

"

,

~

~

,

~

I

t"N

103

800 Z E--;

10 2

0

I

I

I

-100

-200

-300

4OO

-4-00

&EmB/mV Fig. 5. Semilogarithmic plot of the apparent second-order rate constant for the reduction of several viologens by NADPH in the presence of FNR versus the difference between the mid-point redox potentials (pH 8.0) of the N A D P + / N A D P H couple and the viologens. The letters on the curves are identified in Table 1. The solid curve corresponds to the plot of the semi-empirical Marcus equation with VET = 3.2X l0 s and AG # = 4.4 kcal tool- 1.

0 0.0

I

I

I

0.2

0.4

0.6

[NaCI],

t

0.8

I 1.0

1.2

M

Fig. 6. Dependence of the rate of electron transfer from NADPH to oxidized viologens in the presence of FNR on ionic strength • DPV; • MV; • APMV.

184

M.T. Beset al. / Bioelectrochemistry and Bioenergetics 38 (1995) 179-184

viologens, the catalytic rates determined in some cases were similar to the turnover numbers obtained for the reaction using physiological substrates.

Acknowledgments

We are grateful to Dr. G. Tollin for useful discussions and suggestions during the performance of the experimental work and the preparation of the paper, and to Dr C. van Dijk for providing the viologens DAPV and DECV. This work was supported by grants BIO91 1124-C02-01 and BIO91 1124-C02-02 from the Comision Interministerial de Ciencia y Tecnologia, Spain.

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[9] J.C. Hoogvliet, L.C. Lievense, C. van Dijk and C. Veeger, Eur. J. Biochem., 174 (1988) 273. [10] L.A. Summers, The bipyridiniurn herbicides, Academic Press, New York, 1980. [11] R.A. Marcus, Annu. Rev. Phys. Chem., 15 (1964) 155. [12] R.A. Marcus and N. Sutin, Biochim. Biophys. Acta, 811 (1985) 265. [13] G. Tollin, T.E. Meyer and M.A. Cusanovich, Biochim. Biophys. Acta, 853 (1986) 29. [14] G. Zanetti and A. Aliverti, in F. Muller (Ed.), Chemistry and Biochemistry of Flavoenzymes, Vol. II, CRC Press, Boca Raton, FL 1990, p. 305. [15] L.P. Vernon and W.S. Zuagg, J. Biol. Chem., 235 (1960) 2728. [16] M. Avron and A.T. Jagendoff, Arch. Biochem. Biophys., 65 (1956) 475. [17] G. Forti and E. Sturani, Eur. J. Biochem., 3 (1968) 461. [18] J. Sancho, M.L. Peleato, C. Gomez-Moreno and D.E. Edmondson, Arch. Biochem. Biophys., 260 (1988) 200. [19] J.J. Pueyo, C. Gomez-Moreno and S.G. Mayhew, Eur. J. Biochem., 202 (1991) 1065. [20] J.L Pueyo and C. Gomez-Moreno, Prep. Biochem., 21 (1991) 191. [21] D.D. Schlereth and V.M. Fernandez, Biotech. Lett., 11 (1989) 407. [22] E. Katz, A.L. de Lacey, J.L.G. Fierro, J.M. Palacios and V.M. Fernandez, J. Electroanal. Chem., 358 (1993) 247. [23] C.L. Bird and A.T. Kuhn, Chem. Soc. Rev., 10 (1981) 49. [24] I. Willner, J.M. Yang, C. Laaue, J.W. Otvos and M. Calvin, J. Phys. Chem., 85 (1981) 3277. [25] A. Krasna, Photochern. Photobiol., 29 (1978) 267. [26] D.D. Schlereth, Ph.D. Thesis, Universidad Complutense de Madrid, 1990. [27] T. Watanabe and K. Honda, J. Phys. Chem., 86 (1982) 2617. [28] K. Tsukahara and R.G. Wilkins, J. Am. Chem. Soc., 107 (1985) 2632. [29] van Dijk, personal communication. [30] A.R. Marcus, J. Phys. Chem., 72 (1968) 891.