- Email: [email protected]
Electrochemical investigation of binding sites of plantacyanin: blue, copper-containing protein of plants
ELSEVIER
Bioelectrochemistry
and Bioenergetics 40 (I 996) 249-255
Electrochemical investigation of binding sites of plantacyanin: blue, copper-containing protein of plants B.A. Kuznetsov, N.A. Byzova, G.P. Shumakovich, L.E. Mazhorova, A.A. Mutuskin A.N. Bach Insitutr
of’Biochrmi.stry, Russian Academy oj’Sciencrs. Moscow. Russia
Received 24 August 1994; revised 10 January 1996
Abstract
The binding site structure of plantacyanin (plant blue Cu-protein) was studied using voltammetry at the electrodes modified with sulphur-containing compounds (thioglycolic acid, dipyridyl disulfide, cysteamine, f3-mercaptoethanol).The scanning of the functional groups of the modifying layer was used to reveal the most efficient modifications of the electrode and to determine the characterof the binding groups of the protein. A comparison of the electron exchangecharacteristicsof plantacyanin at the different electrodeswith that of the other proteins of the known binding site structure (plastocyanin, cytochrome c, cytochrome ~553, high potential ion-sulfur protein) was carried out. By means of this comparison and also using literary data about plantacyanin tertiary structure, the localization of the protein binding sites and their hydrophobicity were evaluated. Two poles was suggestedto exist with a high permeability to electrons. One of these binding sites is of a high hydrophobicity (4.1-6.7 kJ mol ‘>. The other is hydrophilic and contains several charged amino groups. The problem of how to searchthe partners of plantacyanin in the electron transport chain was discussed. Kry\zord.\: Plantacyanin; Binding sites; Electron transport; ModiIied gold electrode; Cysteamine; Cytochrome ~553
1. Introduction
A blue copper-containing protein of plants, plantacyanin was discovered 20 years ago [l]. Since that time, the amino acid sequence of the protein has been determined [2], X-ray structural analysis of the tertiary structure of the protein has been carried out [3] and many physicochemical properties of the protein have been determined [4,5]. The protein was demonstrated to be localized in plant chloroplasts [4]. However, biological functions of plantacyanin are not yet established although its participation in some reactions has been identified. The possible participation of plantacyain in reactions of PSl 1 was demonstrated [6]. The reactions of plantacyanin with the components of the ferredoxin-NADP-reductase system were studied [7]. A possible function of plantacyanin as component of oxidative system involving peroxidase was considered [8] as well as as an electron carrier in the cycle path of PS1[9]. Nevertheless, until now none of the suggested hypotheses have been developed and they all remain unproven. Plantacyanin has virtually the same redox potential (34OmV) as plastocyanin (370mV) but it is not able to function on the donor side of PSl[lO]. A high positive charge of plantacyanin molecules (pK 10.6) may not allow
them to interact properly with PSI-components on the chloroplast membrane. In addition to the charge, the binding sites play an essential role in providing the necessary interaction and orientation of the protein electron carriers. The binding sites are known to be responsible for the recognition mechanism and the specificity of the electron transfer. The specific interaction of proteins-partners proceeds in an orientation favorable for the electron transfer. Besides, the interaction time becomes more prolonged. which increases the probability of electron tunneling. The mean rate of electron transfer of the protein partners of one organism is two orders of magnitude higher than that of the proteins of different organisms functioning at analogous sites of the chain [l I]. Binding sites provide a specific interaction owing to the interaction of complementary sites of the protein partners, containing charged groups, hydrophobic or hydrophilic surfaces. We believe that the investigation of the binding site of one protein, for example plantacyanin, helps us to draw conclusions about the binding site of its partners. Thus, we need to take into account the structure of the protein binding site in order to determine the position of the protein in the chain of biochemical reactions. The estimation of the structure of the protein binding
0302.4598/96/Sl5,00 Copyright 0 1996 Published by Elsevier Science S.A. All rights reserved. PII SO302-4598(96)05055-6
250
B.A. Kuznet.wu
et al./
Biorlectrochemistry
sites is quite a difficult task. There exists a biochemical method according to which the expected binding groups are screened chemically, then the complex formation and the electron transfer of the modified protein are studied [12]. In this case both a directed synthesis and a determination of the position of protein modification are laborious. In another method, some chemical labels are set to all the external e-amino groups of the lysine residues which are not bound with complementary groups of the protein partner in their complex. After mapping one can determine the lysine groups free from labels which are related to the binding domains [ 131.This method is also time consuming. In this work an electrochemical method was applied to study the binding site structure of plantacyanin. The method is based on known data, obtained over the last lo-15 years, concerning the mechanism of acceleration of the electron exchange of proteins at modified electrodes [ 14,151. The external functional groups in the modifying monolayer were found to be able to orientate protein molecules on the electrode surface and considerably accelerate the electron exchange of proteins at the electrode. The modification of the gold electrode can be carried out with such sulphur-containing compounds as dipyridyl disulfide (DPDS), thioglycolic acid (TGA), cysteamine (CA), B-mercaptoethanol (B-ME). The donor-acceptor bond between sulfur and gold atoms is strong and yields the strict orientation of these compounds on the surface of the gold electrode [ 16-191. After a monolayer of the sulfur compound has formed the -NH=, -COOH, -NH or -OH groups appear respectively on the outer surface of the adsorbed monolayer. The interaction of these groups with the complementary groups on the protein surface makes some superiority of one orientation of protein molecules on the electrode surface. If the orientation is favorable for the electron transfer, the voltammetry peaks of the reversible or quasi-reversible reaction of the electron exchange of the proteins are observed. By scanning the modifying layer one can determine the most efficient modification and hence also the character of the interaction and estimate the groups being in the binding site. If the tertiary structure of the protein is known these data of the binding site can allow determination of its localization. This method is applicable for the proteins in which the direction of both binding and electron tunneling coincide
ml. In this paper the approach described above has been applied to characterize the binding site of plantacyanin. In a previous work the electron transfer was shown to proceed between plantacyanin and ferredoxin in solution [7]. This means that the directions of binding and electron tunneling in each protein coincide or they are close. 2. Experimental Plantacyanin was kindly presented by Drs. R.M. Nalbandian and A.M. Nersisjan from the Institute of Biochem-
and Bioenergetics
40 (19Y6) 249-255
istry, Armenian Academy of Science, Erevan. The protein was isolated from cucumber skin according to the method described elsewhere [ 1,5]. The optical index characterizing the purity of the full oxidized form D,,s/D,g, was equal to 5.9-6.0. The preparation was electrochemically homogenized by electrophoresis in 10% polyacrylamide gel. The concentration of plantacyanin was estimated by its optical density at 597 nm ( Es9, = 2.0 mM- ’ cm- ‘> [6]. Before the experiment, all the plantacyanin samples were purified from the chloride ion traces by a Sephadex G15 column equilibrated with 0.02 M phosphate buffer containing 0.05 M Na,SO,, pH7.6. (Chloride ions interfere in the voltammetry of plantacyanin.) The pH values of the protein solutions were changed by a dialysis over 4h against the analogous buffer with the necessary pH value. The following preparations were used to modify the gold electrode and to prepare the buffer: 4,4’-dipyridyl disulfide (Sigma), B-mercaptoethanol(Ferak), thioglycolic acid (VEB Laborchemie Apolda), KOH (Chemapol), cysteamine, Na,SO, and KH,PO, were recrystallized. All the solutions were prepared using double-distilled water. The modification of the gold electrode was carried out as described elsewhere [16]. The working electrode was a gold wire of 1 mm in diameter sealed in a glass tube. The area of electrode was 0.27cm2. First the electrode was polished with the slurry of 0.3 km alumina in water and washed with double-distilled water. The chemisorption of the modificator was made for 1 min from 1 mM DPDS or B-ME solutions and from 5 mM TGA or CA solutions and their mixture (1: 1) in 0.1 M phosphate buffer, pH 7.0. After washing in a 0.1 M phosphate buffer for 1 min the electrode was ready for the experiment. The investigation of the electron exchange of plantacyanin on the modified gold electrode was carried out by voltammetry. An electrochemical cell was thermostated at 4°C and filled with 0.8 ml of the solution containing 1-2mgmll’ protein in 0.02 M phosphate buffer and 0.05M Na,SO,. Voltammograms were recorded by oscillographic polarograph CLA-03 (Russia), connected with the cell by a three-electrode circuit. The scan rate of the potential was 40 mV s- ’ . Calomel and silver/silver chloride electrodes were used as reference and auxiliary electrodes respectively. The rate constants of the electron exchange of proteins on the electrodes were calculated from the voltammogram parameters according to the method proposed by Nicolson
L211. For calculation of K, an approximation was used made on the basis of the table function of the constant given in Ref. 1211. K, = {24/(ilE,
- 60)}(~oU~~/RT)0.5(cms~‘),
where u is the scan rate of potential (V s-l), D the diffusion coefficient of plantacyanin (cm2 s- ‘), and A E, the potential difference of cathodic and anodic peaks of a voltammogram (mV>.
B.A. Ku;netsou
et aI./
Biorlectrochrmistry
3. Results and discussion Fig. l(a) and (b) shows voltammograms of plantacyanin and plastocyanin in comparison, recorded by using electrodes whose modifications were varied. Plantacyanin is electrochemically active at all the electrodes examined including those modified with DPDS, TGA and CA at which plastocyanin was not active. Thus, the electron exchange of plantacyanin seems to be surprisingly unspecific. For purposes of comparison the qualitative characteristics of the electron exchange of some proteins at the different modified electrodes are shown in Table 1 [ 151.As the tertiary protein structure and the position of the binding sites in these proteins are known [13,22,23] as well as the character of the interaction of functional groups of the sites with the groups of the modifying layer [14,15,20], it becomes possible to estimate the functional groups of plantacyanin involved in the interaction and to evaluate the hydrophobicity of the binding site. Our task proved to be much easier as far as the tertiary structure of plantacyanin is known. Guss et al. [3] found that ligands of the copper atom (a redox center of the molecule) are the imidazole groups of His-84 and 39 which contact directly with the solution. The fastest electron transfer is expected to occur through these imidazole groups. On the globule surface they are surrounded by the uncharged groups: Thr- 12, Phe- 13, Asn35, Met-38 Phe-81, Pro-82 and Ser-87 [3]. There is only
DPDS
I
(b)
Fiw I. Voltammograms of plantacyanin (solid lines) and plastocyanin (dked lines) at the gold electrode modified with DPDS, TGA, CA (a) and modified with a mixture of TGA + CA and P-ME (b). Concentration of both proteins is 3.6mgml-‘, the temperature 4”C, the electrode surface area is 0.27cm’.
cd
Bioenerptio
40 (1996) 249-255
251
Table I Electrochemical activity of plantacyanin and other proteins [ 15,201 at the different modified electrodes Functional groups of the modifying layers
Proteins Pn
OH NH, COOH NH, + COOH PvW COOH+ Mg” the solution
+ + C-j” + + + +
in
PC
Cyt cS53
HIPIP
Cytc
+ -(+)b -
+ -
+ -
+ -
+ -
+
+
+ -
+ + -
Pn, plantacyanin; PC, plastocyanin; Cyt c and Cyt ~553, cytochrome c and c553. a active but at pH < 6 becomes inactive; b it is not active but at pH > 7.6 becomes active.
one charged group Lys-59 which locates a little aside from the domain of His-39 and His-84. The majority of the charged groups locate on the opposite half of the molecule (Lys-20, 22, 69, 72; Arg-23, 25). 3. I. Electrochemistry of plantacyanin and other proteins at the gold electrode modified with TGA Unlike other proteins, plantacyanin is characterized by an intensive electron exchange at the electrode modified with TGA containing carboxyl groups in the modifying layer. Cytochrome c-553 is inactive at this electrode owing to the negatively charged groups being in the binding site: Asp-2, 72; Glu-7, 71 [13,24]. Plastocyanin is inactive because of two reasons: the molecule has a high negative charge and a hydrophobic binding site dominates in the electrochemical reactions 1201. On the contrary, cytochrome c loses its activity at the electrode modified with TGA, owing to a strong electrostatic interaction with the electrode of the whole molecule and of the local charges of the binding sites [15]. Tarlov and Bouden [25] have observed an irreversible binding of cytochrome c on the surface covered with a mono-layer of 16-mercaptohexadecanic acid. Evidently, this strong electrostatic binding should inhibit desorption and the mass exchange of the protein at the electrode. A distribution of positive charges in plantacyanin is more uniform, and unlike those in cytochrome c, they are not concentrated in such great amount in the binding site or in another domain. Therefore, in spite of the high positive charge of plantacyanin molecules, one cannot observe a complete inhibition of the electron exchange at the electrode modified with TGA. The addition of 1 mM MgSO, to the protein electrolyte solution changes the voltammogram (Fig. 2). The potential difference of cathode and anode peaks decreases. This value directly correlates with the constant of electron transfer (K,) and characterizes the degree of irreversibility of the process [21]. The rate constants of electron exchange
252
B.A. Kuzrtsou
et ul. / Bioelrctro~Aenli.~try
Fig. 2. Voltammograms of plantacyanin (I.Omgml~‘) at the gold electrode, modified with TGA; (I) in the electrolyte: 0.05M Na?SO, in 0.02 M phosphate buffer, pH 7.4; (2) as in (I ) but with the addition of I mM MgSO,. The potentials of the peaks are indicated by the dotted line.
of plantacyanin at the electrodes with the different surface modification at several pH values are given in Table 2. At the electrode modified with TGA the rate of electron transfer is an order of magnitude slower than that with other modificators. The constant rate increases twice when adding MgSO,. Thus the orientation of plantacyanin induced with COOH groups of TGA is not quite favorable for the electron exchange. The domain containing the majority of amino groups, i.e. the area at the opposite pole from His-39, 84 is expected to be the most probable place for the interaction of a plantacyanin molecule with the electrode surface. In this case the electron transfer does occur much slower through the matrix of protein. Partial neutralization and recharge of COO- groups by Mg*+ ions apparently cause both an increase in the rotational mobility of adsorbed protein molecules and a decrease of interaction time owing to electrostatic repulsion by improving the mass exchange of protein and electron transfer at electrode. 3.2. Electrochemical behavior of plantacyanin and other proteins at the gold electrode modified with DPDS A high electrochemical activity of the plantacyanin at the electrode modified with DPDS may mean that there are several NH, groups in the protein binding site. Indeed, a fast electrochemical reaction of cytochrome c at this elecTable 2 Rate constants of the electron exchange (K, X IO”cmscyanin on the modified gold electrodes at different pH
40 (19961 249-255
trode is owing to the formation of several hydrogen bonds between protonized E-NH, groups of lysine residues (some of those seven being in the binding site) and nitrogen atoms of pyridine on the electrode surface (pK 4, 5) 1141. There are only two NH, groups (Lys-8, 64) in the hydrophilic site of cytochrome c-553 that apparently cannot provide a reliable orientation of protein molecules for the fast electron exchange. The orientation of the positive pole of plantacyanin to the electrode surface is expected to be efficient for the electron exchange. The hydrogen bonds between DPDS and NH, groups of the protein seem to create more favorable conditions for the rapid electron transfer than the ion bonds COO- and NH3+ at the electrode modified with TGA because the strength of the hydrogen bond is less than that of the ion bond. In this case it may be of importance that the bond strength becomes lower, as in the presence of Mg *+ ions. This results in the increase of the rate of mass exchange and the rotational mobility. Besides the formation of hydrogen bonds, DPDS can take part in hydrophobic interactions. The binding site of the high potential ion-sulfur protein (HIPIP) from purple sulfuric bacteria is highly hydrophobic (8 kJmol-‘1. As DPDS is more hydrophobic than B-ME it can provide a stronger binding of the protein molecules and the limitation of their mass exchange at the electrode. The binding site of plastocyanin dominating in the electrochemical reaction at the electrode modified with B-ME is of a moderate hydrophobicity (5 kJ mall ’ ). The hydrophobicity of the molecule as a whole and of the binding site of plantacyanin is not very high. Therefore, from the strength of binding of plantacyanin molecules on the monolayer of DPDS, hydrophobicity is expected to be moderate and favorable for the fast electron exchange. Indeed the mean hydrophobicity of amino-acid residues surrounding His-84 is equal to 6.7 kJ mol- ’ and that of the whole domain around His-84 and His-39 being 4.9 kJ mol- ’ . There is one amino group of Lys-59 near the binding domain which is able to support the binding and orientation. Thus, there appear to be two pathways for the electron tunneling through the matrix of the plantacyanin molecule at the electrode modified with DPDS. 3.3. Peculiarities of electrochemical behavior of proteins at the gold electrode mod$ed with CA
‘) of planta-
Modification of electrode
PH 6.0
6.6
7.0
7.35
7.6
8.3
CA P-ME CA + TGA TGA TGA+Mg*DPDS
0 6.2 5.0 0.23
3.8 6.2 5.5 0.24
3.1 8.3 4. I 0.23
6.2 7. I 4.5 0.21 0.45 1.3
7.1 6.2 5.0 0.24
6.2 5.5 5.0 0.23
1.34
und Biomrrpdcs
The degree of the ionization of NH, groups on the electrode modified with CA varies in a wide range of pH. This is caused by a strong inhibition of protonization of the new portions of NH, groups by those being protonized earlier [26]. As a result the apparent pK shifts up to 4-5 units. The degree of ionization of amino groups of the modifying layer still changes considerably at pH values 6-8 [20]. Thus on varying the pH value within this interval one can change the density of surface charges on the
B.A. Kuzner.wu
et (11./ Bioelecrrochemistry
and Bioenergerics
40 (1996) 249-255
253
3.4. EfSect of rotational mobility of the protein molecules at the electrode surface on the electrochemical activity
6.0
6.0
pH
Fig. 3. pH-dependence of the height of the voltammetric cathode peak of plantacyanin (solid lines) at the gold electrode modified with 1-A -TGA, 2- n -TGA + CA, 3- 0 -P-ME, CO-CA, and plastocyanin (dotted line) at the electrode modified with CA.
electrode surface and hence the energy of interaction with the charged groups of protein. Fig. 3 shows the dependence of anodic peak current of plantacyanin on pH values at the electrodes modified with CA, TGA, CA + TGA (1: 1) and P-ME. As seen, the change of the peak current takes place only at the electrode modified with CA within a narrow range of the pH value from 6 up to 7. The electron transfer process ceases completely at pH 6. A current decrease could not be caused by conformational changes in the protein as no changes were observed in the electrochemical reaction at the electrodes modified with other compounds. As shown earlier the desorption of the modificator during the experiment is impossible under the condition used [20]. It is suggested that the increase of the density of the positive surface charge at pH 6 can lead only to the electrostatic repulsion from the electrode surface of the plantacyanin molecules bearing a high positive charge. An analogous pH dependence of the peak current of plastocyanin (see Fig. 3) bearing a high negative charge is the result of different effects [20]. As shown, the increase in the density of positive surface charge causes a strong binding of the protein molecules in the orientation, unfavorable for the electron tunneling. Only at low charge density when pH is more than 7.6, a fast electron transfer appears. This effect was investigated and the conclusion was drawn that under this condition the interaction becomes so small that the rotational mobility of the adsorbed protein molecules appears providing the acceleration of their electron exchange at electrode. Thus, the electron transfer of plantacyanin at the electrode modified with CA is rather an unspecific process which occurs when a high rotational mobility of the protein molecules takes place in the adsorbed layer at the electrode surface. At pH < 6, when the surface charge is becoming significantly higher, the interaction time and the amounts of protein molecules adsorbed on the electrode surface considerably decrease. In such conditions the electron transfer ceases.
The rotational mobility of the protein molecules appears to be a determining factor in the acceleration of the electrochemical reactions of different proteins on the electrodes modified with P-ME [ 151. The energy of the hydrogen bond of the hydroxyl group of the P-ME with the ionized carboxyl group and with protonated amino group are equal to the same value of 40 kJ mol- ’ . Hydrophobicity of this compound has an average value; it provides equal possibilities of binding for all parts of the surface of the protein globule that can contain the positively and negatively charged domains as well as hydrophobic and hydrophilic ones. Thus this energetic equality of the protein surface for binding on this modificator appears to cause the rotational mobility of the adsorbed molecules. The data obtained show that the electron exchange of all proteins with the very different structure of the binding sites always occurs at the highest rate on the electrode modified with P-ME [15,20]. As follows from Fig. 3 and Table 2, plantacyanin is not an exception. In our opinion the rotating adsorbed molecule of protein can take an orientation for a short time but one which is one of the more favorable for electron tunneling, much more favorable than some orientation in which the mobility is restricted by a stronger binding. The mixture of TGA and CA forms a mosaic monolayer of the alternating charges of the NH: and COO- groups. Ionization of these groups is expected to be deep or total due to their mutual influence. This modification also creates conditions for the binding of the equal energy but only for the hydrophilic positively and negatively charged areas. This surface has clearly pronounced hydrophilic properties. The reactions of proteins that have hydrophobic binding sites for example of plant plastocyanin and HIPIP from purple sulfuric bacteria are very slow or not registered at all. 3.5. Information binding site
on the hydrophobicity
of the protein
The comparison of rate constants of the electron exchanges on two modified surfaces (hydrophobic of P-ME and hydrophilic of TGA + CA mixture) allows information to be obtained about the hydrophobicity of the protein binding site which provides the optimal orientation for the tunneling of the electron. Table 2 demonstrates the constants of electron exchange rates of plantacyanin on the electrodes with different modificators of the surface. The ratio of the constants of electron transfer on the hydrophilic and hydrophobic electrodes is equal to 0.7. It is interesting to compare this ratio of rate constants to the values of the binding site hydrophobicity for several proteins. The mean value of the hydrophobicity of the site
254
B.A. Ku~net.wo
et cd./ Bioelectrochemistr~
Table 3 Rate constants of the electron exchange of proteins at the electrode hydrophilic (K,, modification TGA + CA) and hydrophobic ( KZ , modik cation P-ME) and average hydrophobicity of the amino acid residue in binding site of proteins (Hy) Protein
K,XlO’
ems-’ HIPIP Plastocyanin site near His-87 Cytochrome c Plantacyanin site near His-39 and His-84
0 0.3 I .24 4.9 (0) a
K>XlO’
ems-’ 2.5 4.9 2.5 6.6 6.6
K,/
K2 0 0.06 0.5 0.7 (0)
Hy Nmol8.0 5.0 4.2 4. I 6.7
’
a Plantacyanin has two hydrophilic sites and the electron path through His-39 appears to be preferable at the hydrophilic surface. At the hydrophobic electrode, both His-39 and His-84 directions are efficient for binding and electron tunneling.
surface was estimated according to the composition of the amino acid residues being on the surface. The value of hydrophobicity of the domain related to one amino acid residue was taken as the measure of the site hydrophobicity. The amino acid hydrophobicity scale of Nozaki and Tanford [27] was used. The analysis of the amino acid residues being on the globule surface in the area of the binding site was carried out according to the known tertiary protein structure using the computer program “Molecular Modeling” [28]. The values of the binding sites hydrophobicity and the ratio of the rate of the electron transfer constants on the hydrophilic (TGA + CA) and hydrophobic (P-ME) electrodes for several proteins are presented in Table 3. The definite opposite course of these values is obvious. The ratio of these constants for plantacyanin is equal to 0.7 and corresponds to the reaction of plantacyanin via a hydrophilic binding site. 3.6. Characteristics of the binding sites of plantacyanin Thus, summing up the information obtained by the comparative analysis of the electrochemical reactions of plantacyanin and proteins at different modified electrodes and taking into consideration the structural peculiarities of plantacyanin, we come to the following conclusion. The fast tunneling of the electrons take place at any rate in two directions or in two orientations of the protein on the electrode. It is confirmed by the following facts. COOH groups catalyze the electron exchange in the modifying layer on the electrode. The electron exchange takes place with the rate by an order lower than that on the other modified surfaces. Apparently, it is connected with the molecule orientation on the electrode. This orientation is formed owing to the local the interactions of the COOH groups situated on the electrode surface with NH, groups of plantacyanin found on the pole opposite to His-39 and His-84. We suppose that the decrease in the rate of the electron transfer in this case is caused by the greater distance of tunneling of the electron path through the
and Bioener~etics
40 (1996) 249-255
protein matrix. Apparently, the highest rates of electron exchange are provided by the orientation of plantacyanin with the side where the copper ligands are situated (imidazole groups of His-39 and His-84) towards the electrode. The mean hydrophobicity of the amino acid residues that are situated near the His-84 imidazole are equal to 6.7 kJ mol- ’ and near His-39 imidazole are equal to 4.1 kJ mol- ‘. The latter value corresponds to the border of hydrophobic and hydrophilic properties and this domain becomes hydrophilic at temperature as low as 4” [29]. This may be a reason why plantacyanin is capable of the fast electron exchange (K, = 1.3 X lo- 3 and 4.5 X 10e3 ems-‘> both on hydrophobic and hydrophilic neutrally charged surfaces (modification DPDS and TGA + CA accordingly). Thus we suppose that at least two poles exist with a great permeability to electrons. On one pole the binding site consists of two parts which are equally uncharged but differ in hydrophobicity. They are both capable of providing the orientation for the effective tunneling of the electrons. On the other pole the binding site is hydrophilic and contains several charged amino groups. This site interacts with the carboxyl group block efficiently enough. 3.7. On possible functions of plantacyanin Presumptive functions of plantacyanin were considered in Section 1 in this paper. It may be that plastocyanin takes part in one or in several of the mentioned reactions: in the reactions of PSI as an electron carrier, in cyclic transport and as an electron acceptor from ferredoxin [6,7], in the reactions of PSI 1[9] and in oxidative processes [8] as well as in other, as yet unknown, processes. Investigation of the biological functions and biochemical behavior of plantacyanin is now in a state of primary accumulation of information. This information is not adequate to put forward a reliable hypothesis on the biological function of plantacyanin. Nevertheless we would like to consider only the reaction that we studied earlier [7] from the point of view of the presented approach according to which the protein partners are sought by the comparison of their binding sites. It was shown that plantacyanin may serve as an electron acceptor from ferredoxin [7]. Ferredoxin was reduced by an electron transport chain of chloroplasts in the light. In the dark this reaction occurs in the presence of NADFH, catalyzed by ferredoxin-NADP-oxidoreductase. The components of this system did not function as an electron donor for plantacyaninin the absence of ferredoxin. The binding site of ferredoxin is known to include a block of three COOH groups [30]. On the contrary, in this work plantacyanin was shown to be reduced and oxidized at the electrode with the COOH groups in the modifying layer at a well observed rate.
B.A. Ku;netsou
er ul. / Bioelectrochemistry
Thus plantacyanin and ferredoxin have, to some degree, complementary binding sites. This is an additional reason to suppose that the electron transfer reaction ferredoxinplantacyanin takes place in chloroplasts. However, supposition that such a hypothesis is of a biological importance demands investigation of many problems. The first of them is to know where plastocyanin is localized in chloroplasts with respect to membrane. If the hypothesis is correct, then the other partneraccepting electron must have a high redox-potential and perhaps a hydrophobic binding site since the hydrophobic site orients the plantacyanin molecules in a position providing the most efficient electron tunneling. Such a second partner may be cytochrome bjS9. Its redox potential is equal to 375mV and the binding site is hydrophobic. Cytochrome b,,, is inaccessible for such a hydrophilic compounds as dithionite and ferredoxin [3 1,321. The validity of the second hypothesis is questioned to a greater extent. We hope, however, that when additional independent data are obtained the results and the approach applied will be more useful for the determination of sequence of the electron carriers in chain around plantacyanin and perhaps in other systems.
References [l] K.A. Markossian, V.Ts. Aikazyan and R.M. Nalbandyan, B&him. Biophys.
Acra., 359 (1974)
47.
[2] M. Murata, G.S.. Begg, F. Lambrou. B. Leslie, R.J. Simpson, H.C. Freeman and F.J. Morgan, Proc. Nurl. Acd. Sci. USA, 79 (1982) 6434. [3] J.H. Guss, E.A. Merritt, R.P. Phyzackerley, B. Hedman, M.Murata, K.O. Hodgson and H.C. Freeman, Science, 241 (1988) 806. [4] V.Ts. Aikazyan and R.M. Nalbandyan, B&him. Biophys. Acta, 667 (1981) 421. [5] A.M. Nersissian, M.A. Babayan, L.K. Sarkissian, E.G. Sarukhanian and R.M. Nalbandian, Biochim. Biophys. Actu, 830 (1985) 195. [6] A.M. Nersissian. Physicochemical and antigen properties of plantacyanin and its biological function. Thesis, Erevan, Armenia, 1990.
und Bwrnergetics
255
40 f 1996) 249-255
[7] A.A. Mutuskin, L.E. Mazhorova, A.M. Nersissian and R.M. Nalbandian, Biokhimiya, 55 (1990) 687 (in Russian). [8] T. Sakurai, H. Okumoto, K. Kawahara and A. Nakahara. FEBS Lrrrs.,
147 (1982)
220.
[9] V.Ts. Haikazyan, The cyclic transfer in the PS I is realized through plantacyanin. Souief - In&m Symp. on regulation rJ~photosynrhrsis, Abstr., Pushchino, 1990, p. 38. [IO] A.A. Mutuskin, L.E. Makovkina, K.V. Pshenova and G.N. Vostroknytova, Biokhimiyu, 42 (1977) 653 (in Russian). Q [l I] J.J. Kulys and V.Y. Razumas, Biocutuly.~is in Electrochemisrry Orpxzic Compounds, Vilnjus, Mokslas, 1983, pp. 58-59. [l2] T. Takabe, H. Ishikawa, S. Niwa and Y. Tanaka, J. Biochem., 96 (1984) 385. [13] R. Rieder and H.R. Bosshard, .I. Bid. Chrm., 25.5 (1980) 4732. [14] J.E. Frew and H.A.O. Hill, Eur. /. Biochrm.. 172 (1988) 261. [15] B.A. Kuznetsov, G.P. Shumakovich, A.A. Mutuskin and L.E. Mazhorova, Dokl. Acud. Nauk USSR, 312 (1990) 1224. [16] L.H. Dubois and R.G. Nuzzo, Ann. Reu. Phys. Chem., 43 (1992) 437. [17] C.D. Bain, E.B. Troghton, Yu-Tai Tao, J. Evall, G.M. Whitesides and R.G. Nuzzo, J. Am. Chem. Sot., 111 (1989) 321-335. 1181 1. Taniguchi, in M.J. Allen, SF. Clary and F.M. Hawkridge teds.), Charge and Field Eflect in Bio.sy.~rem.\, vol. 2, Plenum, 1989, pp. 91-100. [19] Fan Ke-jun, I. Satake, K. Ueda, H. Akutsu and K. Niki, in G. Dryhurst and K. Niki (eds.), Redox Chemistry und Inrer$x?ul Brhauiour of Biological Molecules, Plenum, New York, 1988, p. 125. [20] B.A. Kuznetsov, N.A. Byzova, G.P. Shumakovich, V.K. Gins, L.E. Mazhorova and A.A. Mutuskin, Dokl. Acud. Nuuk SSSR. 329 (1993) 666 (in Russian). [21] R.S. Nicolson, And. Chem., 37 (1965) 1351. [22] J.M. Guss and H.C. Freeman, J. Mol. Bid., 169 (1983) 521. [23] G. Aprahamian and B.A. Feinberg, Biochem.. 20 (1981) 915. [24] D.N. Krogman, Acrcr Physiol. Plunrurum, 8 (1986) 157. [25] M.J. Tarlov and E.F. Bowden, J. Am. Chem. SW., 113 (1991) 1847. [26] D.L. Soloviev and E.U. Kaz, J. Phys. Chem., 64 (1990) 801 (in Russian). [27] Y. Nozaki and C. Tanford, J. Bid. Chem., 246 (1971) 221 I. [28] OUP Molecular Modelling Packqe, Oxford Electronic Publishing, Oxford, 1988. [29] B.A. Kuznetsov, G.P. Shumakovich and N.M. Mestechkina, J. Electroad.
Chem., 248 (1988)
387.
[30] D.B. Knaff and M. Hirasava, Biochim. Biophys. Actu. 12 (1991) 93. [3 I] R.J. Debas, B&him. Biophys. Actu, 1102 (1992) 269-352. [32] L.K. Thompson, A-F. Miller, C.A. Buser, J.C. de Paula and G.W. Brudwig, Biochrm.. 28 (1989) 8048-8056.
Bioelectrochemistry
and Bioenergetics 40 (I 996) 249-255
Electrochemical investigation of binding sites of plantacyanin: blue, copper-containing protein of plants B.A. Kuznetsov, N.A. Byzova, G.P. Shumakovich, L.E. Mazhorova, A.A. Mutuskin A.N. Bach Insitutr
of’Biochrmi.stry, Russian Academy oj’Sciencrs. Moscow. Russia
Received 24 August 1994; revised 10 January 1996
Abstract
The binding site structure of plantacyanin (plant blue Cu-protein) was studied using voltammetry at the electrodes modified with sulphur-containing compounds (thioglycolic acid, dipyridyl disulfide, cysteamine, f3-mercaptoethanol).The scanning of the functional groups of the modifying layer was used to reveal the most efficient modifications of the electrode and to determine the characterof the binding groups of the protein. A comparison of the electron exchangecharacteristicsof plantacyanin at the different electrodeswith that of the other proteins of the known binding site structure (plastocyanin, cytochrome c, cytochrome ~553, high potential ion-sulfur protein) was carried out. By means of this comparison and also using literary data about plantacyanin tertiary structure, the localization of the protein binding sites and their hydrophobicity were evaluated. Two poles was suggestedto exist with a high permeability to electrons. One of these binding sites is of a high hydrophobicity (4.1-6.7 kJ mol ‘>. The other is hydrophilic and contains several charged amino groups. The problem of how to searchthe partners of plantacyanin in the electron transport chain was discussed. Kry\zord.\: Plantacyanin; Binding sites; Electron transport; ModiIied gold electrode; Cysteamine; Cytochrome ~553
1. Introduction
A blue copper-containing protein of plants, plantacyanin was discovered 20 years ago [l]. Since that time, the amino acid sequence of the protein has been determined [2], X-ray structural analysis of the tertiary structure of the protein has been carried out [3] and many physicochemical properties of the protein have been determined [4,5]. The protein was demonstrated to be localized in plant chloroplasts [4]. However, biological functions of plantacyanin are not yet established although its participation in some reactions has been identified. The possible participation of plantacyain in reactions of PSl 1 was demonstrated [6]. The reactions of plantacyanin with the components of the ferredoxin-NADP-reductase system were studied [7]. A possible function of plantacyanin as component of oxidative system involving peroxidase was considered [8] as well as as an electron carrier in the cycle path of PS1[9]. Nevertheless, until now none of the suggested hypotheses have been developed and they all remain unproven. Plantacyanin has virtually the same redox potential (34OmV) as plastocyanin (370mV) but it is not able to function on the donor side of PSl[lO]. A high positive charge of plantacyanin molecules (pK 10.6) may not allow
them to interact properly with PSI-components on the chloroplast membrane. In addition to the charge, the binding sites play an essential role in providing the necessary interaction and orientation of the protein electron carriers. The binding sites are known to be responsible for the recognition mechanism and the specificity of the electron transfer. The specific interaction of proteins-partners proceeds in an orientation favorable for the electron transfer. Besides, the interaction time becomes more prolonged. which increases the probability of electron tunneling. The mean rate of electron transfer of the protein partners of one organism is two orders of magnitude higher than that of the proteins of different organisms functioning at analogous sites of the chain [l I]. Binding sites provide a specific interaction owing to the interaction of complementary sites of the protein partners, containing charged groups, hydrophobic or hydrophilic surfaces. We believe that the investigation of the binding site of one protein, for example plantacyanin, helps us to draw conclusions about the binding site of its partners. Thus, we need to take into account the structure of the protein binding site in order to determine the position of the protein in the chain of biochemical reactions. The estimation of the structure of the protein binding
0302.4598/96/Sl5,00 Copyright 0 1996 Published by Elsevier Science S.A. All rights reserved. PII SO302-4598(96)05055-6
250
B.A. Kuznet.wu
et al./
Biorlectrochemistry
sites is quite a difficult task. There exists a biochemical method according to which the expected binding groups are screened chemically, then the complex formation and the electron transfer of the modified protein are studied [12]. In this case both a directed synthesis and a determination of the position of protein modification are laborious. In another method, some chemical labels are set to all the external e-amino groups of the lysine residues which are not bound with complementary groups of the protein partner in their complex. After mapping one can determine the lysine groups free from labels which are related to the binding domains [ 131.This method is also time consuming. In this work an electrochemical method was applied to study the binding site structure of plantacyanin. The method is based on known data, obtained over the last lo-15 years, concerning the mechanism of acceleration of the electron exchange of proteins at modified electrodes [ 14,151. The external functional groups in the modifying monolayer were found to be able to orientate protein molecules on the electrode surface and considerably accelerate the electron exchange of proteins at the electrode. The modification of the gold electrode can be carried out with such sulphur-containing compounds as dipyridyl disulfide (DPDS), thioglycolic acid (TGA), cysteamine (CA), B-mercaptoethanol (B-ME). The donor-acceptor bond between sulfur and gold atoms is strong and yields the strict orientation of these compounds on the surface of the gold electrode [ 16-191. After a monolayer of the sulfur compound has formed the -NH=, -COOH, -NH or -OH groups appear respectively on the outer surface of the adsorbed monolayer. The interaction of these groups with the complementary groups on the protein surface makes some superiority of one orientation of protein molecules on the electrode surface. If the orientation is favorable for the electron transfer, the voltammetry peaks of the reversible or quasi-reversible reaction of the electron exchange of the proteins are observed. By scanning the modifying layer one can determine the most efficient modification and hence also the character of the interaction and estimate the groups being in the binding site. If the tertiary structure of the protein is known these data of the binding site can allow determination of its localization. This method is applicable for the proteins in which the direction of both binding and electron tunneling coincide
ml. In this paper the approach described above has been applied to characterize the binding site of plantacyanin. In a previous work the electron transfer was shown to proceed between plantacyanin and ferredoxin in solution [7]. This means that the directions of binding and electron tunneling in each protein coincide or they are close. 2. Experimental Plantacyanin was kindly presented by Drs. R.M. Nalbandian and A.M. Nersisjan from the Institute of Biochem-
and Bioenergetics
40 (19Y6) 249-255
istry, Armenian Academy of Science, Erevan. The protein was isolated from cucumber skin according to the method described elsewhere [ 1,5]. The optical index characterizing the purity of the full oxidized form D,,s/D,g, was equal to 5.9-6.0. The preparation was electrochemically homogenized by electrophoresis in 10% polyacrylamide gel. The concentration of plantacyanin was estimated by its optical density at 597 nm ( Es9, = 2.0 mM- ’ cm- ‘> [6]. Before the experiment, all the plantacyanin samples were purified from the chloride ion traces by a Sephadex G15 column equilibrated with 0.02 M phosphate buffer containing 0.05 M Na,SO,, pH7.6. (Chloride ions interfere in the voltammetry of plantacyanin.) The pH values of the protein solutions were changed by a dialysis over 4h against the analogous buffer with the necessary pH value. The following preparations were used to modify the gold electrode and to prepare the buffer: 4,4’-dipyridyl disulfide (Sigma), B-mercaptoethanol(Ferak), thioglycolic acid (VEB Laborchemie Apolda), KOH (Chemapol), cysteamine, Na,SO, and KH,PO, were recrystallized. All the solutions were prepared using double-distilled water. The modification of the gold electrode was carried out as described elsewhere [16]. The working electrode was a gold wire of 1 mm in diameter sealed in a glass tube. The area of electrode was 0.27cm2. First the electrode was polished with the slurry of 0.3 km alumina in water and washed with double-distilled water. The chemisorption of the modificator was made for 1 min from 1 mM DPDS or B-ME solutions and from 5 mM TGA or CA solutions and their mixture (1: 1) in 0.1 M phosphate buffer, pH 7.0. After washing in a 0.1 M phosphate buffer for 1 min the electrode was ready for the experiment. The investigation of the electron exchange of plantacyanin on the modified gold electrode was carried out by voltammetry. An electrochemical cell was thermostated at 4°C and filled with 0.8 ml of the solution containing 1-2mgmll’ protein in 0.02 M phosphate buffer and 0.05M Na,SO,. Voltammograms were recorded by oscillographic polarograph CLA-03 (Russia), connected with the cell by a three-electrode circuit. The scan rate of the potential was 40 mV s- ’ . Calomel and silver/silver chloride electrodes were used as reference and auxiliary electrodes respectively. The rate constants of the electron exchange of proteins on the electrodes were calculated from the voltammogram parameters according to the method proposed by Nicolson
L211. For calculation of K, an approximation was used made on the basis of the table function of the constant given in Ref. 1211. K, = {24/(ilE,
- 60)}(~oU~~/RT)0.5(cms~‘),
where u is the scan rate of potential (V s-l), D the diffusion coefficient of plantacyanin (cm2 s- ‘), and A E, the potential difference of cathodic and anodic peaks of a voltammogram (mV>.
B.A. Ku;netsou
et aI./
Biorlectrochrmistry
3. Results and discussion Fig. l(a) and (b) shows voltammograms of plantacyanin and plastocyanin in comparison, recorded by using electrodes whose modifications were varied. Plantacyanin is electrochemically active at all the electrodes examined including those modified with DPDS, TGA and CA at which plastocyanin was not active. Thus, the electron exchange of plantacyanin seems to be surprisingly unspecific. For purposes of comparison the qualitative characteristics of the electron exchange of some proteins at the different modified electrodes are shown in Table 1 [ 151.As the tertiary protein structure and the position of the binding sites in these proteins are known [13,22,23] as well as the character of the interaction of functional groups of the sites with the groups of the modifying layer [14,15,20], it becomes possible to estimate the functional groups of plantacyanin involved in the interaction and to evaluate the hydrophobicity of the binding site. Our task proved to be much easier as far as the tertiary structure of plantacyanin is known. Guss et al. [3] found that ligands of the copper atom (a redox center of the molecule) are the imidazole groups of His-84 and 39 which contact directly with the solution. The fastest electron transfer is expected to occur through these imidazole groups. On the globule surface they are surrounded by the uncharged groups: Thr- 12, Phe- 13, Asn35, Met-38 Phe-81, Pro-82 and Ser-87 [3]. There is only
DPDS
I
(b)
Fiw I. Voltammograms of plantacyanin (solid lines) and plastocyanin (dked lines) at the gold electrode modified with DPDS, TGA, CA (a) and modified with a mixture of TGA + CA and P-ME (b). Concentration of both proteins is 3.6mgml-‘, the temperature 4”C, the electrode surface area is 0.27cm’.
cd
Bioenerptio
40 (1996) 249-255
251
Table I Electrochemical activity of plantacyanin and other proteins [ 15,201 at the different modified electrodes Functional groups of the modifying layers
Proteins Pn
OH NH, COOH NH, + COOH PvW COOH+ Mg” the solution
+ + C-j” + + + +
in
PC
Cyt cS53
HIPIP
Cytc
+ -(+)b -
+ -
+ -
+ -
+ -
+
+
+ -
+ + -
Pn, plantacyanin; PC, plastocyanin; Cyt c and Cyt ~553, cytochrome c and c553. a active but at pH < 6 becomes inactive; b it is not active but at pH > 7.6 becomes active.
one charged group Lys-59 which locates a little aside from the domain of His-39 and His-84. The majority of the charged groups locate on the opposite half of the molecule (Lys-20, 22, 69, 72; Arg-23, 25). 3. I. Electrochemistry of plantacyanin and other proteins at the gold electrode modified with TGA Unlike other proteins, plantacyanin is characterized by an intensive electron exchange at the electrode modified with TGA containing carboxyl groups in the modifying layer. Cytochrome c-553 is inactive at this electrode owing to the negatively charged groups being in the binding site: Asp-2, 72; Glu-7, 71 [13,24]. Plastocyanin is inactive because of two reasons: the molecule has a high negative charge and a hydrophobic binding site dominates in the electrochemical reactions 1201. On the contrary, cytochrome c loses its activity at the electrode modified with TGA, owing to a strong electrostatic interaction with the electrode of the whole molecule and of the local charges of the binding sites [15]. Tarlov and Bouden [25] have observed an irreversible binding of cytochrome c on the surface covered with a mono-layer of 16-mercaptohexadecanic acid. Evidently, this strong electrostatic binding should inhibit desorption and the mass exchange of the protein at the electrode. A distribution of positive charges in plantacyanin is more uniform, and unlike those in cytochrome c, they are not concentrated in such great amount in the binding site or in another domain. Therefore, in spite of the high positive charge of plantacyanin molecules, one cannot observe a complete inhibition of the electron exchange at the electrode modified with TGA. The addition of 1 mM MgSO, to the protein electrolyte solution changes the voltammogram (Fig. 2). The potential difference of cathode and anode peaks decreases. This value directly correlates with the constant of electron transfer (K,) and characterizes the degree of irreversibility of the process [21]. The rate constants of electron exchange
252
B.A. Kuzrtsou
et ul. / Bioelrctro~Aenli.~try
Fig. 2. Voltammograms of plantacyanin (I.Omgml~‘) at the gold electrode, modified with TGA; (I) in the electrolyte: 0.05M Na?SO, in 0.02 M phosphate buffer, pH 7.4; (2) as in (I ) but with the addition of I mM MgSO,. The potentials of the peaks are indicated by the dotted line.
of plantacyanin at the electrodes with the different surface modification at several pH values are given in Table 2. At the electrode modified with TGA the rate of electron transfer is an order of magnitude slower than that with other modificators. The constant rate increases twice when adding MgSO,. Thus the orientation of plantacyanin induced with COOH groups of TGA is not quite favorable for the electron exchange. The domain containing the majority of amino groups, i.e. the area at the opposite pole from His-39, 84 is expected to be the most probable place for the interaction of a plantacyanin molecule with the electrode surface. In this case the electron transfer does occur much slower through the matrix of protein. Partial neutralization and recharge of COO- groups by Mg*+ ions apparently cause both an increase in the rotational mobility of adsorbed protein molecules and a decrease of interaction time owing to electrostatic repulsion by improving the mass exchange of protein and electron transfer at electrode. 3.2. Electrochemical behavior of plantacyanin and other proteins at the gold electrode modified with DPDS A high electrochemical activity of the plantacyanin at the electrode modified with DPDS may mean that there are several NH, groups in the protein binding site. Indeed, a fast electrochemical reaction of cytochrome c at this elecTable 2 Rate constants of the electron exchange (K, X IO”cmscyanin on the modified gold electrodes at different pH
40 (19961 249-255
trode is owing to the formation of several hydrogen bonds between protonized E-NH, groups of lysine residues (some of those seven being in the binding site) and nitrogen atoms of pyridine on the electrode surface (pK 4, 5) 1141. There are only two NH, groups (Lys-8, 64) in the hydrophilic site of cytochrome c-553 that apparently cannot provide a reliable orientation of protein molecules for the fast electron exchange. The orientation of the positive pole of plantacyanin to the electrode surface is expected to be efficient for the electron exchange. The hydrogen bonds between DPDS and NH, groups of the protein seem to create more favorable conditions for the rapid electron transfer than the ion bonds COO- and NH3+ at the electrode modified with TGA because the strength of the hydrogen bond is less than that of the ion bond. In this case it may be of importance that the bond strength becomes lower, as in the presence of Mg *+ ions. This results in the increase of the rate of mass exchange and the rotational mobility. Besides the formation of hydrogen bonds, DPDS can take part in hydrophobic interactions. The binding site of the high potential ion-sulfur protein (HIPIP) from purple sulfuric bacteria is highly hydrophobic (8 kJmol-‘1. As DPDS is more hydrophobic than B-ME it can provide a stronger binding of the protein molecules and the limitation of their mass exchange at the electrode. The binding site of plastocyanin dominating in the electrochemical reaction at the electrode modified with B-ME is of a moderate hydrophobicity (5 kJ mall ’ ). The hydrophobicity of the molecule as a whole and of the binding site of plantacyanin is not very high. Therefore, from the strength of binding of plantacyanin molecules on the monolayer of DPDS, hydrophobicity is expected to be moderate and favorable for the fast electron exchange. Indeed the mean hydrophobicity of amino-acid residues surrounding His-84 is equal to 6.7 kJ mol- ’ and that of the whole domain around His-84 and His-39 being 4.9 kJ mol- ’ . There is one amino group of Lys-59 near the binding domain which is able to support the binding and orientation. Thus, there appear to be two pathways for the electron tunneling through the matrix of the plantacyanin molecule at the electrode modified with DPDS. 3.3. Peculiarities of electrochemical behavior of proteins at the gold electrode mod$ed with CA
‘) of planta-
Modification of electrode
PH 6.0
6.6
7.0
7.35
7.6
8.3
CA P-ME CA + TGA TGA TGA+Mg*DPDS
0 6.2 5.0 0.23
3.8 6.2 5.5 0.24
3.1 8.3 4. I 0.23
6.2 7. I 4.5 0.21 0.45 1.3
7.1 6.2 5.0 0.24
6.2 5.5 5.0 0.23
1.34
und Biomrrpdcs
The degree of the ionization of NH, groups on the electrode modified with CA varies in a wide range of pH. This is caused by a strong inhibition of protonization of the new portions of NH, groups by those being protonized earlier [26]. As a result the apparent pK shifts up to 4-5 units. The degree of ionization of amino groups of the modifying layer still changes considerably at pH values 6-8 [20]. Thus on varying the pH value within this interval one can change the density of surface charges on the
B.A. Kuzner.wu
et (11./ Bioelecrrochemistry
and Bioenergerics
40 (1996) 249-255
253
3.4. EfSect of rotational mobility of the protein molecules at the electrode surface on the electrochemical activity
6.0
6.0
pH
Fig. 3. pH-dependence of the height of the voltammetric cathode peak of plantacyanin (solid lines) at the gold electrode modified with 1-A -TGA, 2- n -TGA + CA, 3- 0 -P-ME, CO-CA, and plastocyanin (dotted line) at the electrode modified with CA.
electrode surface and hence the energy of interaction with the charged groups of protein. Fig. 3 shows the dependence of anodic peak current of plantacyanin on pH values at the electrodes modified with CA, TGA, CA + TGA (1: 1) and P-ME. As seen, the change of the peak current takes place only at the electrode modified with CA within a narrow range of the pH value from 6 up to 7. The electron transfer process ceases completely at pH 6. A current decrease could not be caused by conformational changes in the protein as no changes were observed in the electrochemical reaction at the electrodes modified with other compounds. As shown earlier the desorption of the modificator during the experiment is impossible under the condition used [20]. It is suggested that the increase of the density of the positive surface charge at pH 6 can lead only to the electrostatic repulsion from the electrode surface of the plantacyanin molecules bearing a high positive charge. An analogous pH dependence of the peak current of plastocyanin (see Fig. 3) bearing a high negative charge is the result of different effects [20]. As shown, the increase in the density of positive surface charge causes a strong binding of the protein molecules in the orientation, unfavorable for the electron tunneling. Only at low charge density when pH is more than 7.6, a fast electron transfer appears. This effect was investigated and the conclusion was drawn that under this condition the interaction becomes so small that the rotational mobility of the adsorbed protein molecules appears providing the acceleration of their electron exchange at electrode. Thus, the electron transfer of plantacyanin at the electrode modified with CA is rather an unspecific process which occurs when a high rotational mobility of the protein molecules takes place in the adsorbed layer at the electrode surface. At pH < 6, when the surface charge is becoming significantly higher, the interaction time and the amounts of protein molecules adsorbed on the electrode surface considerably decrease. In such conditions the electron transfer ceases.
The rotational mobility of the protein molecules appears to be a determining factor in the acceleration of the electrochemical reactions of different proteins on the electrodes modified with P-ME [ 151. The energy of the hydrogen bond of the hydroxyl group of the P-ME with the ionized carboxyl group and with protonated amino group are equal to the same value of 40 kJ mol- ’ . Hydrophobicity of this compound has an average value; it provides equal possibilities of binding for all parts of the surface of the protein globule that can contain the positively and negatively charged domains as well as hydrophobic and hydrophilic ones. Thus this energetic equality of the protein surface for binding on this modificator appears to cause the rotational mobility of the adsorbed molecules. The data obtained show that the electron exchange of all proteins with the very different structure of the binding sites always occurs at the highest rate on the electrode modified with P-ME [15,20]. As follows from Fig. 3 and Table 2, plantacyanin is not an exception. In our opinion the rotating adsorbed molecule of protein can take an orientation for a short time but one which is one of the more favorable for electron tunneling, much more favorable than some orientation in which the mobility is restricted by a stronger binding. The mixture of TGA and CA forms a mosaic monolayer of the alternating charges of the NH: and COO- groups. Ionization of these groups is expected to be deep or total due to their mutual influence. This modification also creates conditions for the binding of the equal energy but only for the hydrophilic positively and negatively charged areas. This surface has clearly pronounced hydrophilic properties. The reactions of proteins that have hydrophobic binding sites for example of plant plastocyanin and HIPIP from purple sulfuric bacteria are very slow or not registered at all. 3.5. Information binding site
on the hydrophobicity
of the protein
The comparison of rate constants of the electron exchanges on two modified surfaces (hydrophobic of P-ME and hydrophilic of TGA + CA mixture) allows information to be obtained about the hydrophobicity of the protein binding site which provides the optimal orientation for the tunneling of the electron. Table 2 demonstrates the constants of electron exchange rates of plantacyanin on the electrodes with different modificators of the surface. The ratio of the constants of electron transfer on the hydrophilic and hydrophobic electrodes is equal to 0.7. It is interesting to compare this ratio of rate constants to the values of the binding site hydrophobicity for several proteins. The mean value of the hydrophobicity of the site
254
B.A. Ku~net.wo
et cd./ Bioelectrochemistr~
Table 3 Rate constants of the electron exchange of proteins at the electrode hydrophilic (K,, modification TGA + CA) and hydrophobic ( KZ , modik cation P-ME) and average hydrophobicity of the amino acid residue in binding site of proteins (Hy) Protein
K,XlO’
ems-’ HIPIP Plastocyanin site near His-87 Cytochrome c Plantacyanin site near His-39 and His-84
0 0.3 I .24 4.9 (0) a
K>XlO’
ems-’ 2.5 4.9 2.5 6.6 6.6
K,/
K2 0 0.06 0.5 0.7 (0)
Hy Nmol8.0 5.0 4.2 4. I 6.7
’
a Plantacyanin has two hydrophilic sites and the electron path through His-39 appears to be preferable at the hydrophilic surface. At the hydrophobic electrode, both His-39 and His-84 directions are efficient for binding and electron tunneling.
surface was estimated according to the composition of the amino acid residues being on the surface. The value of hydrophobicity of the domain related to one amino acid residue was taken as the measure of the site hydrophobicity. The amino acid hydrophobicity scale of Nozaki and Tanford [27] was used. The analysis of the amino acid residues being on the globule surface in the area of the binding site was carried out according to the known tertiary protein structure using the computer program “Molecular Modeling” [28]. The values of the binding sites hydrophobicity and the ratio of the rate of the electron transfer constants on the hydrophilic (TGA + CA) and hydrophobic (P-ME) electrodes for several proteins are presented in Table 3. The definite opposite course of these values is obvious. The ratio of these constants for plantacyanin is equal to 0.7 and corresponds to the reaction of plantacyanin via a hydrophilic binding site. 3.6. Characteristics of the binding sites of plantacyanin Thus, summing up the information obtained by the comparative analysis of the electrochemical reactions of plantacyanin and proteins at different modified electrodes and taking into consideration the structural peculiarities of plantacyanin, we come to the following conclusion. The fast tunneling of the electrons take place at any rate in two directions or in two orientations of the protein on the electrode. It is confirmed by the following facts. COOH groups catalyze the electron exchange in the modifying layer on the electrode. The electron exchange takes place with the rate by an order lower than that on the other modified surfaces. Apparently, it is connected with the molecule orientation on the electrode. This orientation is formed owing to the local the interactions of the COOH groups situated on the electrode surface with NH, groups of plantacyanin found on the pole opposite to His-39 and His-84. We suppose that the decrease in the rate of the electron transfer in this case is caused by the greater distance of tunneling of the electron path through the
and Bioener~etics
40 (1996) 249-255
protein matrix. Apparently, the highest rates of electron exchange are provided by the orientation of plantacyanin with the side where the copper ligands are situated (imidazole groups of His-39 and His-84) towards the electrode. The mean hydrophobicity of the amino acid residues that are situated near the His-84 imidazole are equal to 6.7 kJ mol- ’ and near His-39 imidazole are equal to 4.1 kJ mol- ‘. The latter value corresponds to the border of hydrophobic and hydrophilic properties and this domain becomes hydrophilic at temperature as low as 4” [29]. This may be a reason why plantacyanin is capable of the fast electron exchange (K, = 1.3 X lo- 3 and 4.5 X 10e3 ems-‘> both on hydrophobic and hydrophilic neutrally charged surfaces (modification DPDS and TGA + CA accordingly). Thus we suppose that at least two poles exist with a great permeability to electrons. On one pole the binding site consists of two parts which are equally uncharged but differ in hydrophobicity. They are both capable of providing the orientation for the effective tunneling of the electrons. On the other pole the binding site is hydrophilic and contains several charged amino groups. This site interacts with the carboxyl group block efficiently enough. 3.7. On possible functions of plantacyanin Presumptive functions of plantacyanin were considered in Section 1 in this paper. It may be that plastocyanin takes part in one or in several of the mentioned reactions: in the reactions of PSI as an electron carrier, in cyclic transport and as an electron acceptor from ferredoxin [6,7], in the reactions of PSI 1[9] and in oxidative processes [8] as well as in other, as yet unknown, processes. Investigation of the biological functions and biochemical behavior of plantacyanin is now in a state of primary accumulation of information. This information is not adequate to put forward a reliable hypothesis on the biological function of plantacyanin. Nevertheless we would like to consider only the reaction that we studied earlier [7] from the point of view of the presented approach according to which the protein partners are sought by the comparison of their binding sites. It was shown that plantacyanin may serve as an electron acceptor from ferredoxin [7]. Ferredoxin was reduced by an electron transport chain of chloroplasts in the light. In the dark this reaction occurs in the presence of NADFH, catalyzed by ferredoxin-NADP-oxidoreductase. The components of this system did not function as an electron donor for plantacyaninin the absence of ferredoxin. The binding site of ferredoxin is known to include a block of three COOH groups [30]. On the contrary, in this work plantacyanin was shown to be reduced and oxidized at the electrode with the COOH groups in the modifying layer at a well observed rate.
B.A. Ku;netsou
er ul. / Bioelectrochemistry
Thus plantacyanin and ferredoxin have, to some degree, complementary binding sites. This is an additional reason to suppose that the electron transfer reaction ferredoxinplantacyanin takes place in chloroplasts. However, supposition that such a hypothesis is of a biological importance demands investigation of many problems. The first of them is to know where plastocyanin is localized in chloroplasts with respect to membrane. If the hypothesis is correct, then the other partneraccepting electron must have a high redox-potential and perhaps a hydrophobic binding site since the hydrophobic site orients the plantacyanin molecules in a position providing the most efficient electron tunneling. Such a second partner may be cytochrome bjS9. Its redox potential is equal to 375mV and the binding site is hydrophobic. Cytochrome b,,, is inaccessible for such a hydrophilic compounds as dithionite and ferredoxin [3 1,321. The validity of the second hypothesis is questioned to a greater extent. We hope, however, that when additional independent data are obtained the results and the approach applied will be more useful for the determination of sequence of the electron carriers in chain around plantacyanin and perhaps in other systems.
References [l] K.A. Markossian, V.Ts. Aikazyan and R.M. Nalbandyan, B&him. Biophys.
Acra., 359 (1974)
47.
[2] M. Murata, G.S.. Begg, F. Lambrou. B. Leslie, R.J. Simpson, H.C. Freeman and F.J. Morgan, Proc. Nurl. Acd. Sci. USA, 79 (1982) 6434. [3] J.H. Guss, E.A. Merritt, R.P. Phyzackerley, B. Hedman, M.Murata, K.O. Hodgson and H.C. Freeman, Science, 241 (1988) 806. [4] V.Ts. Aikazyan and R.M. Nalbandyan, B&him. Biophys. Acta, 667 (1981) 421. [5] A.M. Nersissian, M.A. Babayan, L.K. Sarkissian, E.G. Sarukhanian and R.M. Nalbandian, Biochim. Biophys. Actu, 830 (1985) 195. [6] A.M. Nersissian. Physicochemical and antigen properties of plantacyanin and its biological function. Thesis, Erevan, Armenia, 1990.
und Bwrnergetics
255
40 f 1996) 249-255
[7] A.A. Mutuskin, L.E. Mazhorova, A.M. Nersissian and R.M. Nalbandian, Biokhimiya, 55 (1990) 687 (in Russian). [8] T. Sakurai, H. Okumoto, K. Kawahara and A. Nakahara. FEBS Lrrrs.,
147 (1982)
220.
[9] V.Ts. Haikazyan, The cyclic transfer in the PS I is realized through plantacyanin. Souief - In&m Symp. on regulation rJ~photosynrhrsis, Abstr., Pushchino, 1990, p. 38. [IO] A.A. Mutuskin, L.E. Makovkina, K.V. Pshenova and G.N. Vostroknytova, Biokhimiyu, 42 (1977) 653 (in Russian). Q [l I] J.J. Kulys and V.Y. Razumas, Biocutuly.~is in Electrochemisrry Orpxzic Compounds, Vilnjus, Mokslas, 1983, pp. 58-59. [l2] T. Takabe, H. Ishikawa, S. Niwa and Y. Tanaka, J. Biochem., 96 (1984) 385. [13] R. Rieder and H.R. Bosshard, .I. Bid. Chrm., 25.5 (1980) 4732. [14] J.E. Frew and H.A.O. Hill, Eur. /. Biochrm.. 172 (1988) 261. [15] B.A. Kuznetsov, G.P. Shumakovich, A.A. Mutuskin and L.E. Mazhorova, Dokl. Acud. Nauk USSR, 312 (1990) 1224. [16] L.H. Dubois and R.G. Nuzzo, Ann. Reu. Phys. Chem., 43 (1992) 437. [17] C.D. Bain, E.B. Troghton, Yu-Tai Tao, J. Evall, G.M. Whitesides and R.G. Nuzzo, J. Am. Chem. Sot., 111 (1989) 321-335. 1181 1. Taniguchi, in M.J. Allen, SF. Clary and F.M. Hawkridge teds.), Charge and Field Eflect in Bio.sy.~rem.\, vol. 2, Plenum, 1989, pp. 91-100. [19] Fan Ke-jun, I. Satake, K. Ueda, H. Akutsu and K. Niki, in G. Dryhurst and K. Niki (eds.), Redox Chemistry und Inrer$x?ul Brhauiour of Biological Molecules, Plenum, New York, 1988, p. 125. [20] B.A. Kuznetsov, N.A. Byzova, G.P. Shumakovich, V.K. Gins, L.E. Mazhorova and A.A. Mutuskin, Dokl. Acud. Nuuk SSSR. 329 (1993) 666 (in Russian). [21] R.S. Nicolson, And. Chem., 37 (1965) 1351. [22] J.M. Guss and H.C. Freeman, J. Mol. Bid., 169 (1983) 521. [23] G. Aprahamian and B.A. Feinberg, Biochem.. 20 (1981) 915. [24] D.N. Krogman, Acrcr Physiol. Plunrurum, 8 (1986) 157. [25] M.J. Tarlov and E.F. Bowden, J. Am. Chem. SW., 113 (1991) 1847. [26] D.L. Soloviev and E.U. Kaz, J. Phys. Chem., 64 (1990) 801 (in Russian). [27] Y. Nozaki and C. Tanford, J. Bid. Chem., 246 (1971) 221 I. [28] OUP Molecular Modelling Packqe, Oxford Electronic Publishing, Oxford, 1988. [29] B.A. Kuznetsov, G.P. Shumakovich and N.M. Mestechkina, J. Electroad.
Chem., 248 (1988)
387.
[30] D.B. Knaff and M. Hirasava, Biochim. Biophys. Actu. 12 (1991) 93. [3 I] R.J. Debas, B&him. Biophys. Actu, 1102 (1992) 269-352. [32] L.K. Thompson, A-F. Miller, C.A. Buser, J.C. de Paula and G.W. Brudwig, Biochrm.. 28 (1989) 8048-8056.