- Email: [email protected]
Electron transport in biological processes
523
Bioelectrochemistry and Bioenergetics, 17 (1987) 523-534 A section of J. Electroanal. Chem., and constituting Vol. 231 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
988 -
ELECTRON
TRANSPORT
IN BIOLOGICAL
PROCESSES
DEOXYRIBONUCLEIC ACID DEGRADATION AND ITS RELATIONSHIP WITH THE ELECTROCHEMICAL BEHAVIOUR OF HYDROXYQUINONE DERIVATIVES
RICARDO
SCHREBLER
Department DENISE
of Chemistry, B. CIAMPI
*, RAQIJEL Universidad
and MARIA
ARAYA
Catolica de Valparaiso,
DURAN
Institute
de Quimica,
Vniversidade
(Revised
manuscript
received
ARRATIA
Casilla 4059, Valparaiso (Chile)
E. HOFFMANN
Institute de Biologia, Department of Biochemistry, CEP 13.081, Campinas, S.P. (Brazil) NELSON
and ADRIANA
Universidade
Estadual de Campinas,
C. P. 6109,
* Estadual de Campinas,
February
C.P. 6154, CEP 13.081, Campinas,
S.P. (Brazil)
26th 1987)
SUMMARY 2,3,5,6-Tetrahydroxy-2,5-cyclohexadiene-l,4-dione (THCD) autooxidizes under physiological conditions with concomitant production of 5,6-dihydroxy-5-cyclohexane-1,2,3,4-tetrone (DHCT). The (-d[Quinone]/dt)/(d[O;I/dt) ratio indicates mixed one- and two-electron transfer mechanisms. Utilizing a heme group adsorbed on a carbon electrode under anaerobic conditions at pH 6 as model for the respiratory chain, the current peak of the first stage was associated with a net transfer of two electrons and two protons. Experimental redox potentials (U,,,) and the theoretical ionization potential values were indicative that the lowest redox and ionization potentials exhibit higher biological reactivity. This reactivity was tested on DNA transformation.
INTRODUCTION
It is known that hydroxyquinones or quinones play an important role in biological processes, particularly in those where proton and electron transfer processes occur simultaneously [l-3]. A good example of these are redox processes which occur in the mitochondrial membrane of animal cells and in the chloroplast
* To whom correspondence
0302-4598/87/$03.50
should be addressed.
0 1987 Elsevier Sequoia
S.A
524
of plant cells, commonly known as the respiratory chain [3-51. It is believed that there are two chemical species participating in the respiratory chain [3]. One of them is essentially fixed and moves only in synchronization with the mitochondrial internal membrane. In this category, cytochrome and its respective enzymes are included. The other chemical species are formed from those which have certain mobility in the membrane. Ubiquinone in animal cells and plastoquinone, plastocyanin or ferredoxin in plant cells are typical examples [3]. Recently, Slater [4] proposed a double Q cycle for the oxidation of reduced ubiquinone by cytochrome ci. In this cycle the reduced ubiquinone comes from the internal membrane and interacts with cytochrome ci and FeS protein at two oxidation state levels. At the first level the hydroubiquinone is oxidized to semiquinone by cytochrome ci and later to quinone by cytochrome b[558]. During the second stage the hydroubiquinone is oxidized by cytochrome h[566] to semiquinone, which is then oxidized to quinone by cytochrome c,. The oxidized ubiquinone undergoes a diffusional process to the internal membrane where it is again reduced by the cytochrome b[562]. 5,6-Dihydroxy-5-cyclohexane-1,2,3,4-tetrone (DHCT), and 2,3,5,6tetrahydroxy2,5-cyclohexadiene-1,4-dione (THCD) exhibit antitrypanosomal [6] and antiviral activity [7,8] and inhibition of glyoxylase I [9]. The latter selective inhibition could
THCC pH Dependent Process
02 Dependent Process
(1)
02 1em
2 e-
I I
(11) One or two electron reactlons
H202
,’ :
:
I THCD
I \
(III)
( DHCT?
hS b DHCT
Scheme 1.
Tautomerlzatlons
GeneratIon of excited states upon free radical Interaction
525
explain its carcinostatic activity by preventing metabolism of a-ketoaldehydes in tumor cells. Presumably, all of these effects are related to redox processes and can be rationalized reasonably on the basis of MNDO calculations [lo]. These properties led us to study the autooxidation of these substituted hydroxyquinones and to establish a relationship between these processes and the biological effects induced by these substances. We observed that the acyl-ene-diol moiety of THCD does indeed undergo autooxidation under physiological conditions with the generation of the intermediate ene-diol radical, superoxide radical anion and hydroxyl radicals, the final product being hydrogen peroxide and DHCT [ll]. In the light of the available information we suggested a reaction scheme which explains the general behaviour of quinones in uiuo. There are still some unknown aspects of these processes. For example, the degree of interaction of the quinones with cytochrome, especially with its heme groups or other functional groups responsible for the electronic charge transfer in the respiratory chain, are still unknown. The reaction mechanisms involved in these processes as well as the detection and identification of the intermediates are other aspects of interest. In order to establish the redox mechanism for these types of species, electrochemical studies on several structurally similar quinones have been performed. Fleury et al. [12-141 studied DHCT as a model of ubiquinone in a wide range of pH values, The acid-base characteristics as well as its electrochemical behaviour (reversibility and characterization of the reduction products under different conditions) were established. They also concluded that this process occurs in the sequence: DHCT + THCD + HHB with two electrons in each step. Other quinones which have been studied are 1,4_naphthoquinones [15,16], benzoquinone [16,17], anthraquinone [17], etc. Ubiquinone has been studied only in non-aqueous solvents [18-221. The objective of this work was to investigate the electrochemical behaviour of DHCT at an electrode interphase constituted by a modified pyrolytic carbon electrode with hemin molecules adsorbed on its surface. Also, the importance of the intermediates in biological reactivity was studied. EXPERIMENTAL
A water-jacketed Pyrex cell thermostatted at 25 k O.l”C was used. A pyrolytic carbon electrode (Le carbone Lorraine) was constructed using a teflon support with an apparent area of 0.04 cm2. The reference electrode was an aqueous saturated KC1 calomel electrode (s.c.e.) located in a compartment with a Luggin-Haber capillary. The counter electrode was a platinum electrode with an apparent area of 10 cm2. Treatment of the carbon electrode was as follows: First the electrode was polished mechanically with a C Si-800 paper, then washed with triply distilled water and dried with filter paper. Then the electrode was either immersed into the cell (Type A electrode) or immersed in a Hemin 0.1 mM basic solution (1 M KOH) for about 12 hours. Then it was washed and immersed in the electrolytic cell (Type B electrode). The electrode was then maintained for 1 minute at a potential q. = 0.1 V and a repetitive triangular potential scan was applied at rates from 0.001 V/s to
526
0.060 V/s while recording the response of the third cycle in each case. Electrochemical measurements were conducted in McIlvaine buffer solution [12] at 3 < pH < 8.2. The ionic strength was kept constant with 0.5 M KCl. The quinone concentration was varied from 0 to 1 mM. The solutions were previously deareated with nitrogen for 1 hour and the measurements were carried out under a continuous stream of nitrogen. The hemin chloride was from Alderich. All other reagents were analytical purity from Merck. The electrochemical measurements were performed with a Tacussel PRT 30-01 potentiostat and with a Tacussel GSTP 3 function generator coupled with a Watanabe X-Y recorder, Model WX4 301. The MNDO procedure (modified neglect of diatomic overlap) for the ionization potential calculations, is a semiempirical version of the NDDO approximation (neglect of diatomic differential overlap) of the Roothaan-Hell SCF-MO [lo]. Crystallographic data were utilized for THCD; the geometries of the other molecules were based on standard molecular geometrical parameters. Planarity of the molecular skeleton was assumed. All the calculations were performed on the DEC-PDP-10 Computer of the Computational Center at Unicamp. The oxygen uptake measurements were conducted using a Clark type oxygen electrode. The appearance of the superoxide radical anion was measured by the nitroblue tetrazolium chloride (Sigma) test [ll]. The kinetics of the disappearance of THCD was carried out at 480 nm in a Zeiss DMR-10 Spectrophotometer. DNA degradation was carried out following the method of Hoffmann and Meneghini [23]. Chemiluminescence experiments were carried out using a Liquid Scintillation Counter, Beckmann LS 1OOc, as described previously [ll].
RESULTS
AND
DISCIJSSION
The biological reactivity order is THCD > DHCT. The redox potentials lJi,*) at pH 6.5 [24] and the Au,,,[13] of DHCT and THCD are given in Table 1. The order of increasing redox potential is THCD < DHCT. Table 1 also shows typical values calculated by the MNDO method [lo] for some thermodynamic and spectroscopic properties of the two molecules.
TABLE
1
A HP and ionazation
potential
Property
A HP (kcal/mol) Ionization potential $2
A$2
m
(mv)
for quinone
derivatives
Systems
eV n
DHCT
THCD
Ref.
- 165.53 10.34 +0.32 - 600
- 191.88 9.90 +0.30 - 100
This work This work 24 13
521
From Table 1 the exothermicity is higher for THCD than for DHCT. THCD also showed the lowest ionization potential, which is in accord with its lower redox potential and higher biological reactivity. These results clearly suggest that all of these processes are related to redox processes and that the effect is probably directly related to radical intermediate formation. THCD autooxidizes under physiological conditions as found by oxygen consumption with concomitant production of DHCT (not shown). There is a good correlation between the rate of disappearance of THCD (- d[Q]/dt) and that of the appearance of the superoxide radical anion as measured by the nitroblue tetrazolium chloride test (d[O;]/dt). The parameter K, introduced by Yamazaki [25] to determine the extent of one- or two-electron transfer reactions in oxidoreductive enzyme reactions, can be applied to our system. Under our conditions the ratio between the disappearance of the THCD and the appearance of O;, (-d[Q]/dt)/(d[OJ/dt), results in a K value of 1.2. This K value is indicative of a mixed one- and two-electron transfer mechanism in this autooxidation process. Scheme 1 shows the possible mechanism. In order to understand better these results under physiological conditions we studied the behaviour of DHCT under electrochemical conditions and the reversibility of the DHCT + THCD process. In order to simulate better the physiological conditions, experiments were carried out with a carbon electrode with and without a heme group adsorbed on the electrode surface as an oxidant instead of oxygen. This allowed us to analyse the intermediates in the redox process better than in the presence of oxygen. Figures la and lc show the voltammetric response of the two interphases that have been studied within a potential range of U, = 0.1 V to 17, = - 0.9 V. In this figure it can be observed that the adsorbed heme group exhibits a highly reversible process and that this is independent of the number of cycles applied to the electrode. This behaviour is indicative of the high stability of the hemin adsorbed on the surface. The signal results exclusively from the transformation of Fe(II1) to Fe(I1) by the potential variation. Figures lb and Id show the voltammetric signal of a 0.1 mM DHCT solution on these two cathodic current peaks (I, and II,) and two conjugated anodic current peaks (1, and 11,) which appear when the potential is reversed. The limiting current observed in both cases at potentials lower than UP, II, is indicative of a diffusion controlled process. A careful analysis of these curves shows that there are differences in the electrochemical behaviour of DHCT at these two interphases. First, when the modified electrode was used, a shift of the cathodic peak toward more positive potential values and a shift of the anodic peak to more negative potential values, was found. When the base current is eliminated there is a net increase of the associated current for the processes. These facts are indicative that the electrochemical process in the presence of an adsorbed heme group on the electrode is thermodynamically and kinetically more favourable than in its absence. Figure 2 shows the voltammetric response of DHCT at the two electrode surfaces at different pH values. The behaviour agrees with that of other quinones under
528
Fig. 1. Potentiodynamic U/I responses of different interphases, recorded at 0.010 V/s at 25 o C. (a) C McIlvaine buffer, pH 8.0; (b) C McIlvaine buffer, pH 8.0+0.1 mM DHCT; (c) C [Heme](,,,, McIlvaine McIlvaine buffer, pH 8.0 +O.l mM DHCT. buffer, pH 8.0; and (d) C[Heme](,,,,
similar conditions [16]. However, the waves not only exhibit a potential shift, they also loose their electrochemical reversibility as the pH is decreased. In contrast, reversibility was not lost even at pH 3.0 when electrode B was used. The appearance of waves at pH G 5.5 is indicative that the electroactive species present are not the same. Fleury et al. [12-141 attributed this behaviour to the protonation of DHCT. In order to insure that the electroactive species was always the same, all experiments were done within the pH range of 6 to 8, where only the most deprotonated species exist. In Fig. 3 the most likely species present in this range are pointed out. This is obvious from the pK, values of 3.5 and 4.5, respectively. Another interesting aspect observed in Fig. 2 is the potential of the redox processes of DHCT in relation to the potential of the process associated with the heme group. At pH G 5.5 DHCT is reduced before the heme group. However, at pH > 6.5 the redox processes of the quinones occur at the same potential or at lower values than the reduction of the heme group. Figure 4 shows the changes of the potential values of cathodic peak II, with the perturbation rate at different pH values. These curves show that the slope of the Tafel plot for the electrochemical process goes to zero, even at pH 6.5, when the process takes place on the modified surface. This indicates that the process is more reversible electrochemically. On the contrary, when the unmodified carbon surface is used, the process exhibits a slope larger than zero in all cases, even at pH 8.2. The same behaviour can be deduced from the plot of log l(rc, uer3u.s log u (Fig. 5). In all of these cases, a value of 0.5 for the slope (a log I,/8 log U) was found, indicating
529
-1 0
-0 5
c/(V)
0.0
I
-1.0
I
-0.5
1 I U(V)
I
0.0
,
I
N
Fig. 2. pH influence on the response of 1 mM 25 o C. (a) C McIlvaine buffer; (b) C [Heme](,,,, (5) 3.0; and (. -. - .) without DHCT.
DHCT at the two interphases recorded at 0.010 V/s at McIlvaine buffer at pH: (1) 8.0; (2) 6.5; (3) 5.5; (4) 4.5;
that the process has diffusional character. However, the current peaks are larger in the C [heme] (ads) interphase than in the carbon alone. Similar results, as shown in Figs. la and lb for peak II,, have been found in relation to the cathodic current
OHC-?* 2H,O
GHCT-2
Fig. 3. Species which are produced
DHCT-'
in the pH range 6-8
Fig. 4. Potential variation, -) Electrode A; ( ---)
,,J , 0.6
Up, of cathodic peak IIc with the scan rate in 1 mM DHCT solution. electrode B. (0, n) pH 8.2; (0,O) pH 7.5; and (0, 0) pH 6.5.
, Lag v/(mV 5-O 10
(-
-
, 14
Fig. 5. Current variation, Zp, of peak IIc with the scan rate. (0) Electrode B; (0) electrode value of the signal obtained with 1 mM DHCT in McIlvaine buffer at 6.5 Q pH $ 8.2.
A. Average
531 TABLE Variation
2 of the peak-to-peak
PH
8.0 7.5 6.5 5.5 4.5 3.0
potential
difference
of the redox pairs with pH a Au,, II (v)
Au,> 1 (v) C [Heme]
C
C [heme]
C
0.040 0.030 0.036 0.030 0.040 0.044
0.090 0.140 0.140 0.140 irrev. irrev.
0.040 0.030 0.036 0.030 0.040 0.065
90 140 190 irrev. irrev. irrev.
a Scan rate = 0.010 V/s.
peak lc and in the oxidation peaks II, and I,. For each peak a linear relationship was found between UP and log u. A slope of zero was obtained for the C [Heme] (ads) interphase, and one larger than zero for the C lsolution interphase. Again, the current peaks are always larger when a type B electrode is used. The plot of log ZP versus log u gives a straight line with a slope of 0.5. Table 2 shows the variation of the anodic and cathodic peak-to-peak potential difference, as a function of pH. This parameter again indicates that the reversibility is maintained until pH = 6.0 in the presence of the heme group. In its absence the electrochemical reduction of DHCT decreases. All of these experimental facts support the catalytic reduction of DHCT by the heme group. This is in agreement with other electrochemical studies of quinones [26], and with the known catalytic properties of porphyrinic groups [27-301. A possible model for the reduction of these polyquinones could be similar to the one proposed by Fleury et al. [12-141 for pH higher than 7.0, Fig.3. However, this mechanism must be modified to include heme group participation. This group probably acts simply as a charge transfer agent in a stepwise manner. It can act as an electron donor or as an electron acceptor. This is due to the rapid charge transfer process of the Fe(II1) [Heme]/Fe(II) [Heme] couple. Taking into account all of these facts, the formal reaction scheme proposed is the following: (I) Before reaching the initial potential, both the heme group as well as the DHCT are at a high oxidation state and could be associated: C[Heme](III)
+ DHCT2-
(II) During then DHCT:
the potential
C[Heme(III)
. . DHCT’-]
+ C[Heme(III).
. . DHCT2-]
scan (at pH 6.0) the heme group + e- + Hf + C[Heme(II)
is reduced
. . . DHCT-]
(I) first and
(2)
. . . DHCT-] + e- + H + C[Heme(III) . . . THCD2-] (3) This scheme is associated with current peak I (Fig. 1) and a net transfer of two electrons and two protons. Coulometric analysis within the potential range of peak I shows a two-electron process. This is in agreement with the results of Fleury et al. [12-141. C[Heme(II)
532
(III) In this potential region the heme group is reduced This continues the reduction process of THCD to HHB. C[Heme(III)
. . . THCD2-]
C[Heme(II)...THCD’-] C[Heme(III).
+ e- + H+ + C[ Heme(I1).
almost
. . THCD’-]
+eP+H++C[Heme(III)...HHB2-]
. . HHB2-]
instantaneously.
(4) (5)
+ eP + 2 H+ + C[Heme(II)]
+ HHB
(6)
This reaction sequence is associated with current peak II (Fig. l), with a net transfer of three electrons and four protons. An important observation was the lack of dismutation of the intermediate found by Fleury [15] at pH higher than 7.0. This is probably due to the association between THCD and the heme group, which should shift this secondary reaction to lower pH values. On the anodic sweep, the reactions associated with the cathodic current peaks II, and I, should be the reverse of the ones described above, since the redox processes involved are very reversible. Taking into account the relative potentials of the processes led us to propose a scheme where the quinone participates uia a concentration gradient. In the alkaline range the spontaneous reaction should be: 2 [Heme Fe(III)]
-t HHB + 2 [Heme Fe(II)]
In the acid range the spontaneous 2 [ Heme Fe(II)]
+ DHCT
reaction
+ THCD’should
+ 2 H+ + 2 [ Heme Fe(III)]
+ 2 H+
(7)
be: + THCD
(8)
As can be noted from the reaction scheme, where DHCT is transformed to THCT [equations (2) and (3)] in a reversible way there is a net transfer of two electrons. This agrees with the K values measured by a non-electrochemical method.
Fig. 6. Chemiluminescence concentration.
of the autooxidation
of THCD in 0.1 M phosphate buffer as a function of
533
Fig. 7. Stern-Volmer plot for the quenching of the chemiluminescence of the autooxidation THCD (0) and 1.6 mM DHCT @) in phosphate buffer at different DNA concentrations.
of 2 mM
The easy transformation of DHCT to THCD as shown by the electrochemical analysis and from our previous autooxidation studies suggests that it could be possible to react these quinones with some biological targets. We selected deoxyribonucleic acid (DNA) as the target for the interaction of the radical intermediates formed during the reversible transformation of DHCT to THCD [ll]. Figure 6 shows the linearity of the chemiluminescence process with a 0.25 mM THCD concentration with an efficiency of 6 X 10 photons ss’ (mM THCD)-‘. Figure 7 shows Stern-Volmer plots for THCD and DHCT using DNA as quencher [31]. Since the autoo.xidation of these quinones results in a non-specific production of activated oxygen species, the quenching is a good indication of the reactivity of these species with DNA. It was previously shown that THCD is more reactive than DHCT in biological systems and the observation that the ratio of Ksv for THCD and DHCT is 1.6 is a corroboration that the redox index and the activity parameter correlate quite well. The same order of reactivity upon analysis of DNA cleavage was observed (not shown). In summary, THCD is a very reactive compound toward biological targets and this behaviour is related directly to its redox properties. These facts could explain the biological activities observed for these quinones in previous work.
ACKNOWLEDGEMENTS
This work was supported by Direction General de Investigation de la Universidad Catolica de Valparaiso-Chile, Binational OAS Programme (Brazil-Chile) Binational CNPq-CONICYT Programme (Brazil-Chile), FAPESP, FINEP, and UNESCO Programme. N.D. thanks the Guggenheim Foundation (U.S.A.) and DAAD Foundation (Germany) for fellowships. We also thank Dr. Luis Echegoyen of the University of Miami (U.S.A.) for a critical reading of the manuscript, and Dr. Enrique Cadenas from Linkijping University (Sweden) for some preliminary experiments on quinone chemiluminescence.
534 REFERENCES 1 R. Bentley and I.M. Cambell in The Chemistry of the Quinoid Compounds, S. Patai (Editor), Wiley, Londen, 1974, Part II, Ch. 13, p. 638. 2 H.J. Harmon and V.G. Struble, Biochemistry, 22 (1983) 4394. 3 J.M. Anderson, and B. Anderson, Trends Biochem. Sci., 7 (1982) 288. 4 E.C. Slater, Trends B&hem. Sci., 8 (1983) 234. 5 L.I. Hochstein and S.E. Cronin, Can. J. Microbial., 30 (1984) 572. 6 M.M.T. Khan and A.E. Martell, J. Amer. Chem. Sot., 89 (1967) 4176. 7 M.Ya. Kraft, V.V. Katishkina, G.N. Pershin and N.S. Bogdanova, Farmakol. Toksikol., 29 (1966) 59. 8 H. Mueke and M. Sproessig, Arch. Exp. Veterinaermed., 21 (1967) 307. 9 K.T. Douglas and I.N. Negri, FEBS Lett., 106 (1979) 393. 10 0. Machuca, M.E. Hoffmann and N. Duran, Actual. Fis. Quim. Org. (Brazil), 3 (1984) 241. 11 M.E. Hoffmann, D.B. Ciampi and N. Duran, Experientia. 43 (1987) 217. 12 M.B. Fleury and G. Molle, Electrochim. Acta, 20 (1975) 951. 13 M.B. Fleury, Electrochim. Acta, 21 (1976) 913. 14 J. Moiroux, D. Escourrou and M.B. Fleury, J. Electroanal. Chem., 116 (1980) 333. 15 V. Glezer, J. Stradins. J. Freimanis and L. Baider, Electrochim. Acta, 28 (1983) 87. 16 S.I. Bailey and I.M. Ritchie, Electrochim. Acta, 30 (1985) 3. 17 J.Q. Chambers, in The Chemistry of the Quinoid Compounds, S. Patai (Editor), Wiley, London, 1974, Part II, Ch. 14, p. 737. 18 G. Cauquis and G. Marbach, Experientia Suppl., (1971) 205. 19 M. Shaushall, R.H. Ghathban and S.M. Ali, Stud. Biophys., 103 (1984) 201. 20 M. Shaushall, R.H. Ghathban and S.M. AIi, Stud. Biophys., 103 (1984) 209. 21 M. Shaushall and K.H. Hassau, Stud. Biophys.. 105 (1985) 59. 22 M. Shaushall and K.H. Hassau. Stud. Biophys., 105 (1985) 65. 23 M.E. Hoffmann and R. Meneghini, Photochem. Photobiol., 30 (1979) 151. 24 P. Souchay and F. Jatibonet, J. Chim. Phys., 49 (1952) C108. 25 I. Yamazaki in Free Radicals in Biology, W.A. Pryor (Editor), Academic Press, New York, 1982, Vol. 3, p. 83. 26 C. Degrand, L. Roullier, L.L. Miller and B. Zinger, J. Electroanal. Chem., 178 (1984) 101. 27 J. Zagal, P. Brindra and E. Yeager, J. Electrochem. Sot., 127 (1980) 1506. 28 M. Yamana, R. Darby and R.E. White, Electrochim. Acta, 29 (1984) 329. 29 X.Y. Wang, R. Motekait and A.E. Martell, Inorg. Chem., 23 (1984) 271. 30 CF. Kolpin and H.S. Swofford, Jr., Anal. Chem., 50 (1978) 920. 31 N. Duran, ST. Farias-Furtado, A. Faljoni-Alario, A. Campa, J.E. Brunet and J. Freer, J. Photochem., 25 (1984) 285.
Bioelectrochemistry and Bioenergetics, 17 (1987) 523-534 A section of J. Electroanal. Chem., and constituting Vol. 231 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
988 -
ELECTRON
TRANSPORT
IN BIOLOGICAL
PROCESSES
DEOXYRIBONUCLEIC ACID DEGRADATION AND ITS RELATIONSHIP WITH THE ELECTROCHEMICAL BEHAVIOUR OF HYDROXYQUINONE DERIVATIVES
RICARDO
SCHREBLER
Department DENISE
of Chemistry, B. CIAMPI
*, RAQIJEL Universidad
and MARIA
ARAYA
Catolica de Valparaiso,
DURAN
Institute
de Quimica,
Vniversidade
(Revised
manuscript
received
ARRATIA
Casilla 4059, Valparaiso (Chile)
E. HOFFMANN
Institute de Biologia, Department of Biochemistry, CEP 13.081, Campinas, S.P. (Brazil) NELSON
and ADRIANA
Universidade
Estadual de Campinas,
C. P. 6109,
* Estadual de Campinas,
February
C.P. 6154, CEP 13.081, Campinas,
S.P. (Brazil)
26th 1987)
SUMMARY 2,3,5,6-Tetrahydroxy-2,5-cyclohexadiene-l,4-dione (THCD) autooxidizes under physiological conditions with concomitant production of 5,6-dihydroxy-5-cyclohexane-1,2,3,4-tetrone (DHCT). The (-d[Quinone]/dt)/(d[O;I/dt) ratio indicates mixed one- and two-electron transfer mechanisms. Utilizing a heme group adsorbed on a carbon electrode under anaerobic conditions at pH 6 as model for the respiratory chain, the current peak of the first stage was associated with a net transfer of two electrons and two protons. Experimental redox potentials (U,,,) and the theoretical ionization potential values were indicative that the lowest redox and ionization potentials exhibit higher biological reactivity. This reactivity was tested on DNA transformation.
INTRODUCTION
It is known that hydroxyquinones or quinones play an important role in biological processes, particularly in those where proton and electron transfer processes occur simultaneously [l-3]. A good example of these are redox processes which occur in the mitochondrial membrane of animal cells and in the chloroplast
* To whom correspondence
0302-4598/87/$03.50
should be addressed.
0 1987 Elsevier Sequoia
S.A
524
of plant cells, commonly known as the respiratory chain [3-51. It is believed that there are two chemical species participating in the respiratory chain [3]. One of them is essentially fixed and moves only in synchronization with the mitochondrial internal membrane. In this category, cytochrome and its respective enzymes are included. The other chemical species are formed from those which have certain mobility in the membrane. Ubiquinone in animal cells and plastoquinone, plastocyanin or ferredoxin in plant cells are typical examples [3]. Recently, Slater [4] proposed a double Q cycle for the oxidation of reduced ubiquinone by cytochrome ci. In this cycle the reduced ubiquinone comes from the internal membrane and interacts with cytochrome ci and FeS protein at two oxidation state levels. At the first level the hydroubiquinone is oxidized to semiquinone by cytochrome ci and later to quinone by cytochrome b[558]. During the second stage the hydroubiquinone is oxidized by cytochrome h[566] to semiquinone, which is then oxidized to quinone by cytochrome c,. The oxidized ubiquinone undergoes a diffusional process to the internal membrane where it is again reduced by the cytochrome b[562]. 5,6-Dihydroxy-5-cyclohexane-1,2,3,4-tetrone (DHCT), and 2,3,5,6tetrahydroxy2,5-cyclohexadiene-1,4-dione (THCD) exhibit antitrypanosomal [6] and antiviral activity [7,8] and inhibition of glyoxylase I [9]. The latter selective inhibition could
THCC pH Dependent Process
02 Dependent Process
(1)
02 1em
2 e-
I I
(11) One or two electron reactlons
H202
,’ :
:
I THCD
I \
(III)
( DHCT?
hS b DHCT
Scheme 1.
Tautomerlzatlons
GeneratIon of excited states upon free radical Interaction
525
explain its carcinostatic activity by preventing metabolism of a-ketoaldehydes in tumor cells. Presumably, all of these effects are related to redox processes and can be rationalized reasonably on the basis of MNDO calculations [lo]. These properties led us to study the autooxidation of these substituted hydroxyquinones and to establish a relationship between these processes and the biological effects induced by these substances. We observed that the acyl-ene-diol moiety of THCD does indeed undergo autooxidation under physiological conditions with the generation of the intermediate ene-diol radical, superoxide radical anion and hydroxyl radicals, the final product being hydrogen peroxide and DHCT [ll]. In the light of the available information we suggested a reaction scheme which explains the general behaviour of quinones in uiuo. There are still some unknown aspects of these processes. For example, the degree of interaction of the quinones with cytochrome, especially with its heme groups or other functional groups responsible for the electronic charge transfer in the respiratory chain, are still unknown. The reaction mechanisms involved in these processes as well as the detection and identification of the intermediates are other aspects of interest. In order to establish the redox mechanism for these types of species, electrochemical studies on several structurally similar quinones have been performed. Fleury et al. [12-141 studied DHCT as a model of ubiquinone in a wide range of pH values, The acid-base characteristics as well as its electrochemical behaviour (reversibility and characterization of the reduction products under different conditions) were established. They also concluded that this process occurs in the sequence: DHCT + THCD + HHB with two electrons in each step. Other quinones which have been studied are 1,4_naphthoquinones [15,16], benzoquinone [16,17], anthraquinone [17], etc. Ubiquinone has been studied only in non-aqueous solvents [18-221. The objective of this work was to investigate the electrochemical behaviour of DHCT at an electrode interphase constituted by a modified pyrolytic carbon electrode with hemin molecules adsorbed on its surface. Also, the importance of the intermediates in biological reactivity was studied. EXPERIMENTAL
A water-jacketed Pyrex cell thermostatted at 25 k O.l”C was used. A pyrolytic carbon electrode (Le carbone Lorraine) was constructed using a teflon support with an apparent area of 0.04 cm2. The reference electrode was an aqueous saturated KC1 calomel electrode (s.c.e.) located in a compartment with a Luggin-Haber capillary. The counter electrode was a platinum electrode with an apparent area of 10 cm2. Treatment of the carbon electrode was as follows: First the electrode was polished mechanically with a C Si-800 paper, then washed with triply distilled water and dried with filter paper. Then the electrode was either immersed into the cell (Type A electrode) or immersed in a Hemin 0.1 mM basic solution (1 M KOH) for about 12 hours. Then it was washed and immersed in the electrolytic cell (Type B electrode). The electrode was then maintained for 1 minute at a potential q. = 0.1 V and a repetitive triangular potential scan was applied at rates from 0.001 V/s to
526
0.060 V/s while recording the response of the third cycle in each case. Electrochemical measurements were conducted in McIlvaine buffer solution [12] at 3 < pH < 8.2. The ionic strength was kept constant with 0.5 M KCl. The quinone concentration was varied from 0 to 1 mM. The solutions were previously deareated with nitrogen for 1 hour and the measurements were carried out under a continuous stream of nitrogen. The hemin chloride was from Alderich. All other reagents were analytical purity from Merck. The electrochemical measurements were performed with a Tacussel PRT 30-01 potentiostat and with a Tacussel GSTP 3 function generator coupled with a Watanabe X-Y recorder, Model WX4 301. The MNDO procedure (modified neglect of diatomic overlap) for the ionization potential calculations, is a semiempirical version of the NDDO approximation (neglect of diatomic differential overlap) of the Roothaan-Hell SCF-MO [lo]. Crystallographic data were utilized for THCD; the geometries of the other molecules were based on standard molecular geometrical parameters. Planarity of the molecular skeleton was assumed. All the calculations were performed on the DEC-PDP-10 Computer of the Computational Center at Unicamp. The oxygen uptake measurements were conducted using a Clark type oxygen electrode. The appearance of the superoxide radical anion was measured by the nitroblue tetrazolium chloride (Sigma) test [ll]. The kinetics of the disappearance of THCD was carried out at 480 nm in a Zeiss DMR-10 Spectrophotometer. DNA degradation was carried out following the method of Hoffmann and Meneghini [23]. Chemiluminescence experiments were carried out using a Liquid Scintillation Counter, Beckmann LS 1OOc, as described previously [ll].
RESULTS
AND
DISCIJSSION
The biological reactivity order is THCD > DHCT. The redox potentials lJi,*) at pH 6.5 [24] and the Au,,,[13] of DHCT and THCD are given in Table 1. The order of increasing redox potential is THCD < DHCT. Table 1 also shows typical values calculated by the MNDO method [lo] for some thermodynamic and spectroscopic properties of the two molecules.
TABLE
1
A HP and ionazation
potential
Property
A HP (kcal/mol) Ionization potential $2
A$2
m
(mv)
for quinone
derivatives
Systems
eV n
DHCT
THCD
Ref.
- 165.53 10.34 +0.32 - 600
- 191.88 9.90 +0.30 - 100
This work This work 24 13
521
From Table 1 the exothermicity is higher for THCD than for DHCT. THCD also showed the lowest ionization potential, which is in accord with its lower redox potential and higher biological reactivity. These results clearly suggest that all of these processes are related to redox processes and that the effect is probably directly related to radical intermediate formation. THCD autooxidizes under physiological conditions as found by oxygen consumption with concomitant production of DHCT (not shown). There is a good correlation between the rate of disappearance of THCD (- d[Q]/dt) and that of the appearance of the superoxide radical anion as measured by the nitroblue tetrazolium chloride test (d[O;]/dt). The parameter K, introduced by Yamazaki [25] to determine the extent of one- or two-electron transfer reactions in oxidoreductive enzyme reactions, can be applied to our system. Under our conditions the ratio between the disappearance of the THCD and the appearance of O;, (-d[Q]/dt)/(d[OJ/dt), results in a K value of 1.2. This K value is indicative of a mixed one- and two-electron transfer mechanism in this autooxidation process. Scheme 1 shows the possible mechanism. In order to understand better these results under physiological conditions we studied the behaviour of DHCT under electrochemical conditions and the reversibility of the DHCT + THCD process. In order to simulate better the physiological conditions, experiments were carried out with a carbon electrode with and without a heme group adsorbed on the electrode surface as an oxidant instead of oxygen. This allowed us to analyse the intermediates in the redox process better than in the presence of oxygen. Figures la and lc show the voltammetric response of the two interphases that have been studied within a potential range of U, = 0.1 V to 17, = - 0.9 V. In this figure it can be observed that the adsorbed heme group exhibits a highly reversible process and that this is independent of the number of cycles applied to the electrode. This behaviour is indicative of the high stability of the hemin adsorbed on the surface. The signal results exclusively from the transformation of Fe(II1) to Fe(I1) by the potential variation. Figures lb and Id show the voltammetric signal of a 0.1 mM DHCT solution on these two cathodic current peaks (I, and II,) and two conjugated anodic current peaks (1, and 11,) which appear when the potential is reversed. The limiting current observed in both cases at potentials lower than UP, II, is indicative of a diffusion controlled process. A careful analysis of these curves shows that there are differences in the electrochemical behaviour of DHCT at these two interphases. First, when the modified electrode was used, a shift of the cathodic peak toward more positive potential values and a shift of the anodic peak to more negative potential values, was found. When the base current is eliminated there is a net increase of the associated current for the processes. These facts are indicative that the electrochemical process in the presence of an adsorbed heme group on the electrode is thermodynamically and kinetically more favourable than in its absence. Figure 2 shows the voltammetric response of DHCT at the two electrode surfaces at different pH values. The behaviour agrees with that of other quinones under
528
Fig. 1. Potentiodynamic U/I responses of different interphases, recorded at 0.010 V/s at 25 o C. (a) C McIlvaine buffer, pH 8.0; (b) C McIlvaine buffer, pH 8.0+0.1 mM DHCT; (c) C [Heme](,,,, McIlvaine McIlvaine buffer, pH 8.0 +O.l mM DHCT. buffer, pH 8.0; and (d) C[Heme](,,,,
similar conditions [16]. However, the waves not only exhibit a potential shift, they also loose their electrochemical reversibility as the pH is decreased. In contrast, reversibility was not lost even at pH 3.0 when electrode B was used. The appearance of waves at pH G 5.5 is indicative that the electroactive species present are not the same. Fleury et al. [12-141 attributed this behaviour to the protonation of DHCT. In order to insure that the electroactive species was always the same, all experiments were done within the pH range of 6 to 8, where only the most deprotonated species exist. In Fig. 3 the most likely species present in this range are pointed out. This is obvious from the pK, values of 3.5 and 4.5, respectively. Another interesting aspect observed in Fig. 2 is the potential of the redox processes of DHCT in relation to the potential of the process associated with the heme group. At pH G 5.5 DHCT is reduced before the heme group. However, at pH > 6.5 the redox processes of the quinones occur at the same potential or at lower values than the reduction of the heme group. Figure 4 shows the changes of the potential values of cathodic peak II, with the perturbation rate at different pH values. These curves show that the slope of the Tafel plot for the electrochemical process goes to zero, even at pH 6.5, when the process takes place on the modified surface. This indicates that the process is more reversible electrochemically. On the contrary, when the unmodified carbon surface is used, the process exhibits a slope larger than zero in all cases, even at pH 8.2. The same behaviour can be deduced from the plot of log l(rc, uer3u.s log u (Fig. 5). In all of these cases, a value of 0.5 for the slope (a log I,/8 log U) was found, indicating
529
-1 0
-0 5
c/(V)
0.0
I
-1.0
I
-0.5
1 I U(V)
I
0.0
,
I
N
Fig. 2. pH influence on the response of 1 mM 25 o C. (a) C McIlvaine buffer; (b) C [Heme](,,,, (5) 3.0; and (. -. - .) without DHCT.
DHCT at the two interphases recorded at 0.010 V/s at McIlvaine buffer at pH: (1) 8.0; (2) 6.5; (3) 5.5; (4) 4.5;
that the process has diffusional character. However, the current peaks are larger in the C [heme] (ads) interphase than in the carbon alone. Similar results, as shown in Figs. la and lb for peak II,, have been found in relation to the cathodic current
OHC-?* 2H,O
GHCT-2
Fig. 3. Species which are produced
DHCT-'
in the pH range 6-8
Fig. 4. Potential variation, -) Electrode A; ( ---)
,,J , 0.6
Up, of cathodic peak IIc with the scan rate in 1 mM DHCT solution. electrode B. (0, n) pH 8.2; (0,O) pH 7.5; and (0, 0) pH 6.5.
, Lag v/(mV 5-O 10
(-
-
, 14
Fig. 5. Current variation, Zp, of peak IIc with the scan rate. (0) Electrode B; (0) electrode value of the signal obtained with 1 mM DHCT in McIlvaine buffer at 6.5 Q pH $ 8.2.
A. Average
531 TABLE Variation
2 of the peak-to-peak
PH
8.0 7.5 6.5 5.5 4.5 3.0
potential
difference
of the redox pairs with pH a Au,, II (v)
Au,> 1 (v) C [Heme]
C
C [heme]
C
0.040 0.030 0.036 0.030 0.040 0.044
0.090 0.140 0.140 0.140 irrev. irrev.
0.040 0.030 0.036 0.030 0.040 0.065
90 140 190 irrev. irrev. irrev.
a Scan rate = 0.010 V/s.
peak lc and in the oxidation peaks II, and I,. For each peak a linear relationship was found between UP and log u. A slope of zero was obtained for the C [Heme] (ads) interphase, and one larger than zero for the C lsolution interphase. Again, the current peaks are always larger when a type B electrode is used. The plot of log ZP versus log u gives a straight line with a slope of 0.5. Table 2 shows the variation of the anodic and cathodic peak-to-peak potential difference, as a function of pH. This parameter again indicates that the reversibility is maintained until pH = 6.0 in the presence of the heme group. In its absence the electrochemical reduction of DHCT decreases. All of these experimental facts support the catalytic reduction of DHCT by the heme group. This is in agreement with other electrochemical studies of quinones [26], and with the known catalytic properties of porphyrinic groups [27-301. A possible model for the reduction of these polyquinones could be similar to the one proposed by Fleury et al. [12-141 for pH higher than 7.0, Fig.3. However, this mechanism must be modified to include heme group participation. This group probably acts simply as a charge transfer agent in a stepwise manner. It can act as an electron donor or as an electron acceptor. This is due to the rapid charge transfer process of the Fe(II1) [Heme]/Fe(II) [Heme] couple. Taking into account all of these facts, the formal reaction scheme proposed is the following: (I) Before reaching the initial potential, both the heme group as well as the DHCT are at a high oxidation state and could be associated: C[Heme](III)
+ DHCT2-
(II) During then DHCT:
the potential
C[Heme(III)
. . DHCT’-]
+ C[Heme(III).
. . DHCT2-]
scan (at pH 6.0) the heme group + e- + Hf + C[Heme(II)
is reduced
. . . DHCT-]
(I) first and
(2)
. . . DHCT-] + e- + H + C[Heme(III) . . . THCD2-] (3) This scheme is associated with current peak I (Fig. 1) and a net transfer of two electrons and two protons. Coulometric analysis within the potential range of peak I shows a two-electron process. This is in agreement with the results of Fleury et al. [12-141. C[Heme(II)
532
(III) In this potential region the heme group is reduced This continues the reduction process of THCD to HHB. C[Heme(III)
. . . THCD2-]
C[Heme(II)...THCD’-] C[Heme(III).
+ e- + H+ + C[ Heme(I1).
almost
. . THCD’-]
+eP+H++C[Heme(III)...HHB2-]
. . HHB2-]
instantaneously.
(4) (5)
+ eP + 2 H+ + C[Heme(II)]
+ HHB
(6)
This reaction sequence is associated with current peak II (Fig. l), with a net transfer of three electrons and four protons. An important observation was the lack of dismutation of the intermediate found by Fleury [15] at pH higher than 7.0. This is probably due to the association between THCD and the heme group, which should shift this secondary reaction to lower pH values. On the anodic sweep, the reactions associated with the cathodic current peaks II, and I, should be the reverse of the ones described above, since the redox processes involved are very reversible. Taking into account the relative potentials of the processes led us to propose a scheme where the quinone participates uia a concentration gradient. In the alkaline range the spontaneous reaction should be: 2 [Heme Fe(III)]
-t HHB + 2 [Heme Fe(II)]
In the acid range the spontaneous 2 [ Heme Fe(II)]
+ DHCT
reaction
+ THCD’should
+ 2 H+ + 2 [ Heme Fe(III)]
+ 2 H+
(7)
be: + THCD
(8)
As can be noted from the reaction scheme, where DHCT is transformed to THCT [equations (2) and (3)] in a reversible way there is a net transfer of two electrons. This agrees with the K values measured by a non-electrochemical method.
Fig. 6. Chemiluminescence concentration.
of the autooxidation
of THCD in 0.1 M phosphate buffer as a function of
533
Fig. 7. Stern-Volmer plot for the quenching of the chemiluminescence of the autooxidation THCD (0) and 1.6 mM DHCT @) in phosphate buffer at different DNA concentrations.
of 2 mM
The easy transformation of DHCT to THCD as shown by the electrochemical analysis and from our previous autooxidation studies suggests that it could be possible to react these quinones with some biological targets. We selected deoxyribonucleic acid (DNA) as the target for the interaction of the radical intermediates formed during the reversible transformation of DHCT to THCD [ll]. Figure 6 shows the linearity of the chemiluminescence process with a 0.25 mM THCD concentration with an efficiency of 6 X 10 photons ss’ (mM THCD)-‘. Figure 7 shows Stern-Volmer plots for THCD and DHCT using DNA as quencher [31]. Since the autoo.xidation of these quinones results in a non-specific production of activated oxygen species, the quenching is a good indication of the reactivity of these species with DNA. It was previously shown that THCD is more reactive than DHCT in biological systems and the observation that the ratio of Ksv for THCD and DHCT is 1.6 is a corroboration that the redox index and the activity parameter correlate quite well. The same order of reactivity upon analysis of DNA cleavage was observed (not shown). In summary, THCD is a very reactive compound toward biological targets and this behaviour is related directly to its redox properties. These facts could explain the biological activities observed for these quinones in previous work.
ACKNOWLEDGEMENTS
This work was supported by Direction General de Investigation de la Universidad Catolica de Valparaiso-Chile, Binational OAS Programme (Brazil-Chile) Binational CNPq-CONICYT Programme (Brazil-Chile), FAPESP, FINEP, and UNESCO Programme. N.D. thanks the Guggenheim Foundation (U.S.A.) and DAAD Foundation (Germany) for fellowships. We also thank Dr. Luis Echegoyen of the University of Miami (U.S.A.) for a critical reading of the manuscript, and Dr. Enrique Cadenas from Linkijping University (Sweden) for some preliminary experiments on quinone chemiluminescence.
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