Electron-Transfer from Cytochrome c to Ascorbate Oxidase and Its Type 2 Copper-Depleted Derivatives

Takeshi Sakurai

College of Liberal Arts and Sciences, Kanazawa University, Kanazawa, Ishikuwa, Japan

ABSTRACT

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting cucumber ascorbate oxidase as functions of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 820 M-’ s-’ in 0.2 M phosphate buffer at pH 6.0 and 25°C. The activation parameters were estimated to be AH* = 5 kJ mol-’ and AS* = - 188 Jmol-* K-l. The rate constants increased with decreasing buffer concentration, indicating that the electron-transfer from cytochrome c to ascorbate oxidase is.realizd by the local electrostatic interaction between them in spite of the reaction between positively charged proteins. Reactions of type 2 copper- depleted ascorbate oxidase whose type 3 coppers were in the reduced or oxidized form indicated that the type 1 copper site accepts an electron from cytochrome c. The reaction rate was remarkably increassd with decreasing pH for both the native enzyme and derivatives. Further, on addition of hexametaphosphate anion the rate of the electron-transfer decreased because the association of both proteins to realize the electron-transfer was inhibited due to a change in distribution of the local charge on the protein surface(s).

INTRODUCTION

In order to clarify the highly directional electron-transfer reactions with which metalloproteins concern, special attention has been focused on biological pro- cesses such as respiration and photosynthesis 111. Factors which govern the rate of these processes are distance, intervening medium, orientation, and redox potential difference between the donor and acceptor [21. At the initial stage of the biological electron-transfer, the redox couple are required to properly recognize each other. Although the reactions between natural redox partners

Address reprint requests and correspondence to: Dr. Takeshi Sakurai, College of Liberal Arts and Sciences, Kanazawa University, Kakuma, Ishikawa 920-11, Japan.

Joumal of Inorganic Biochemisby, 55,193-202 (1993) 193 0 1994 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/94/$7.00

194 T. Sakurai

are goals of the studies, the reactions between metalloproteins which are not the intrinsic redox partners and the reactions involving a small oxidant or redundant have also been very informative in exploring the mechanism of the biological electron-transfer process and origins of specificity and efficiency in macro- molecule recognition [3]. In line with this, the reactions between cytochrome c and blue copper proteins, plastocyanin and azurin, have been examined by kinetic methods [4]. Studies on these electron mediators have also been per- formed by flash-photolysis and pulse radiolysis [5] and by direct electrochemistry El.

Ascorbate oxidase is a multicopper oxidase widely distributed in higher plants and microorganisms [7]. The active site of this enzyme has been shown to be composed of a set of four copper ions, one type 1 copper (blue copper), one type 2 copper (one-blue copper) and two type 3 coppers (ESR undetectable coppers). Recently, the x-ray crystallography has revealed the spatial ayangement of these copper sites [8]. The type 1 copper is separated ca. 12-13 A from the unusual trinuclear center constructed by the type 2 and type 3 coppers. Studies on ascorbate oxidase have been relatively few comparing to those of other multi- copper oxidases, lactase, and ceruloplasmin. Many of the studies have focused in defining the structure of the complex active site [7, 91.

The reaction of ascorbate oxidase begins with the electron-transfer from the substrate to the type 1 copper site, being followed by the intramolecu- lar electron-transfer between the type 1 copper and the trinuclear center [lo]. However, kinetic information of this enzyme is extremely limited [ill, although the refined crystal structure study showed the putative binding site for a substrate near the type 1 copper [8b].

In the present study, we report the electron-transfer reaction between ascor- bate oxidase and cytochrome c in comparison with the reaction between lactase and cytochrome c [12]. The reason cytochrome c was used as the electron donor is that the property of this heme protein has been examined thoroughly by spectroscopic and electrochemical methods, and its reactions with various reagents have been well-examined by kinetic methods [13].

MATERIALS AND METHODS

Materials

Ascorbate oxidase was isolated from cucumber peelings according to the litera-

ture D41. A,,-,/A,,, as the purity index was 25. The gel electrophoresis gave a single band. The type 2 copper-depleted derivative whose type 3 coppers were in the reduced form was prepared according to the literature [SC]. The oxidation of the type 3 copper was performed by treating the derivative with H,Oz (X 10) 1151. Excess H,O, was dialyzed off. Horse heart cytochrome c was purchased from Sigma Chemical Co., Inc. (type VI, 99% purity), and was dialyxed against the buffer solution before use. Protein concentrations were determined based on the molar absorptivities, eao7 = 10,000 for ascorbate oxidase and l ss0 = 27,500 for reduced cytochrome c. Potassium phosphate buffer was used throughout the experiments. All chemicals were of the highest quality available and were used without further purification. Water was deionized and distilled.

ELEXXRON TRANSFER OF ASCORBATE OKIDASE 195

Apparatus and Procedures

Cytochrome c in the reduced form was prepared by treating the resting protein with a small amount of dithionite and by dializing it against a buffer solution under N,. Reduced cytochrome c was soon used for kinetic measurements in order to avoid its gradual autooxidation under air. Kinetic experiments were performed in a 1 cm path length cell on an Otsuka Denshi MCPD-1000 spectrophotometer with photodiode array detector. Absorption spectra in the range 300-700 nm were monitored at 1 set intervals for 3 min and the data of the absorbance change of reduced cytochrome c at 550 nm were stored in a desktop computer. Decrease of absorbance at the wavelength obeyed first-order kinetics at the early stage of the reaction. The margins of error (reproducibility) were approximately f 20%.

RESULTS

Ionic Strength Dependence of the Electron-Transfer Reactions between Cytochrome c and Ascorbate Oxldase

For the kinetic measurements, 2- to lo-fold excess of cytochrome c to ascorbate oxidase was used. Four sets of reactions were performed in 0.2, 0.1, 0.004, and 0.008 M phosphate buffer at pH 6.0 and 2s”C.

The electron-transfer reaction was followed by a second-order process

and the reaction rate could be obtained by the equation

-d[Cyt,,l/dt = ko&ytredl = k[AO+,JICjltred

where kobs is the first-order rate constant and k is the second-order rate constant for the oxidation of reduced cytochrome c by ascorbate oxidase. The rate constants kobs depending on the concentration of cytochrome c are displayed in the inset in Figure 1. Log values of the averaged second-order rates for four sets of data at each buffer concentration were plotted against the square root of the ionic strength in Figure 1. The ionic strengths were calculated from ionization constants of phosphoric acid (p&r, PK,,, and pK,, values are 2.12, 7.21, and 12.32, respectively) and the resulting species distribution curve. The Debye-Hiickel’s limiting law was applied to interpret the fact that the reaction proceeded faster with decreasing buffer concentration (see Discussion).

Temperature Dependence of the Electron-Transfer Reactions between Cytochrome c and Ascorbate Oxidase

Kinetic measurements have been performed at 7, 13, 19, and 25°C in 0.2 M phosphate buffer at pH 6.0 (Fig. 2). The reaction rates were scarcely indepen- dent of temperature, and the thermodynamic parameters, AH* = 5 kJmol_’ and AS* = -188 Jmol-’ K-l were obtained according to the linear least- squares fit.

196 T. Sakurai

3.0 -

I

0 0.2 o-k JT Oa6 Oa8

FIGURE 1. Plots of the second-order rate constants for cytochrome c oxidation by ascorbate oxidase versus square root of the ionic strength of phosphate buffer (pH 6.0, WT). Inset: Kinetics of the electron-transfer from reduced cytochrome c to ascorbate oxidase in 0.2 CO), 0.1 (01, 0.04 (A ), and 0.007 M (0) phosphate buffer (ascorbate oxidase concentration was 0.5 PM.

I I I I I I I I

3.2 3.3 3.4 3.5 3.6

103/l FIGURE 2. Eyrins plots of the electron-transfer rate from reduced cytochrome c (3 PM) to ascorbate oxidase (0.5 PM) WC, pH 6.0, 0.2 M phosphate buffer).

ELECTRON TRANSFER OF ASCORBATE OXIDASE 197

pH Dependence of the Electron-Transfer Reactions between Cytochrome c and Ascorbate Oxidase

We investigated the pH dependence of the reactions of native ascorbate oxidase and type 2 copper-depleted derivatives in 0.2 M phosphate buffer at 25°C. Figure 3 shows that the rate of the reactions markedly increases with decreasing pH for both the native enzyme and derivatives.

Effect of Hexametaphosphate Anion on the Electron-Transfer Reactions between Cytochrome c and Ascorbate Oxidase

One or more hexametaphosphate anion(s) has been revealed to bind to the basic patch(s) of cytochrome c surface surrounding the exposed heme edge [16]. Since this polyanion is expected to affect the electron-transfer process by modulating the manner of the interaction between cytochrome c and ascorbate oxidase, the experiments were performed by using cytochrome c previously equilibrated with hexametaphosphate. Figure 4 shows that an increasing amount of the polyanion effectively inhibits the electron-transfer process.

DISCUSSION

Ascorbate oxidase, as well as lactase [121, appeared to serve as an electron acceptor from reduced cytochrome c. The second-order rate constants for the oxidation of reduced cytochrome c by ascorbate oxidase and lactase were k = 820 M-’ s-i and 125 M-l s-’ [12], respectively, in 0.2 M phosphate buffer at pH 6.0 and 25°C.

TZDT3deoxy \

1 ,)L& Native

5 6

PH

FIGURE 3. Effect of pH on the second-order rate of the oxidation of cytochrome c (5.7 PM) by ascorbate oxidase and its derivatives (0.7 PM) (WT, 0.2 M phosphate buffer).

198 i? Sakumi

1000

800 4

600 rl I u)

,+ I E: - 400 x

200

0

Hexametaphosphate/ Cytochrome c

0 1 2

I I 0 5 10

[Hexametaphosphatel (PM)

FIGURE 4. Effect of hexametaphosphate on the second-order rate constant of the oxidation of cytoehrome c (5.3 PM) by ascorbate oxidase (0.7 PM) W’C, 0.008 mM phosphate buffer).

The result of the ionic strength dependence (Fig. 1) shows that an electro- static interaction operates in the association which allows the electron-transfer between cytochrome c and ascorbate oxidase. According to the Debye-Hiickel’s limiting law, the following simplified relation is retained for the reaction of the charged reactants (k, rate constant; I, ionic strength, Z, and Z,, charges of reactants A and B):

log k = l.O182fiZ,Z,.

Although the proper function of ionic strength should be fi/<afi + 11, where a is the parameter for radius, it is very near to fi. From the slope of the line in Figure 1, which was determined according to the linear least-squares fit, Z,Z, = - 13 was obtained. This shows that an electrostatic interaction between two proteins allows the electron-transfer.

However, both proteins are positively charged at pH 6: the total charges of cytochrome c and ascorbate oxidase are +7.5 [13] and +8 [17l, respectively. Therefore, it appears that the association, which realizes the electron-transfer

ELECTRON TRANSFER OF ASCORBATE OXIDASE 199

between both proteins, is favored by a local electrostatic interaction. A similar situation has also been observed in the electron-transfer reaction between lactase and cytochrome c, in which Z,Z, was -0.9 (the net charge of lactase is +30 at pH 6) [12]. Reactions between the proteins with the same signed total charge have been reported to be favored at a high ionic strength [181. Therefore, the present result suggests that an association of the biological reactants should be highly directional. The rate of the electron-transfer between positively charged cytochrome c and cytochrome f has been reported to be much slower (4 x 10e7 M- ’ s- ‘1 [19]. On the other hand, the rate of the electron-transfer between oppositely charged cytochrome f and plastocyanin has been reported to occur at a much faster rate (4.5 X lo7 M- ’ sml) [4b].

The activation enthalpy was 5 kJ mol-’ (Fig. 2). Such a small value has been reported for the intramolecular electron-transfer of modified axurin, in which Ru was bound to the remote His-83 [20]. The activation enthalpy is smaller than that for the reaction between plastocyanin and cytochrome f (36 kJ mol-‘)[4bl. The activation entropy (- 188 Km01 -’ K- ‘1 is similar to that observed for the reaction between lactase and cytochrome c (- 153 Jmol-’ K-‘)[12], whilst it is far from that observed for the reaction between plastocyanin and cytochrome f (20 Jmol-’ K-l) [4b].

The thermodynamic driving force for the electron-transfer between cyto- chrome c and ascorbate oxidase is relatively strong: the redox potentials of both proteins are 250 mV [22] and 350 mV [231, respectively, and AE” is 100 mV, being large enough for a much faster process. However, the rather slow rate might come from that the appropriate association of both proteins, a pre- requisite to realize the electron-transfer, is not necessarily easy in spite of favorable AE”.

The fact above will be due to that net charges of both proteins are positive. In addition, the type 1 copper site in ascorbate oxidase is deeply buried inside protein molecules as has been shown by x-ray crystallography 181. While an imidaxole group of a ligand histidine is exposed to exterior solvent in the case of blue copper proteins [23], the corresponding imidaxole group is located at the bottom of the possible binding pocket for the organic substrate in the case of ascorbate oxidase [8b]. Consequently, the porphyrin0 edge of cytochrome c, the exit of an electron, will be separated more than 10 A from the type 1 ccpper of ascorbate oxidase, although we cannot estimate the exact distance at present. The electron-transfer becomes unfavorable with increasing distance between the donor and acceptor [21]. Further, the intervening groups and medium are also very important. No favorable group for the electron-transfer seems to be present in the substrate binding pocket of ascorbate oxidase. In the case of lactase, AE”, the thermodynamic driving force of the electron-transfer was 130 mV [12], the slightly larger value than the present case of ascorbate oxidase. However, the reaction had been relatively slower (vide supra). Although we do not have structural information to discuss this further, it is certain that the electron- transfer from cytochrome c to multicopper oxidases is more favorable in the case of ascorbate oxidase than in the case of lactase as far as the electron-transfer from cytochrome c is concerned.

The site which accepts an electron is the type 1 copper. It can be ascertained that the type 2 copper-depleted enzyme, in which the type 3 coppers are in the reduced form, showed the apparent ability to accept an electron from cytochrome

200 T. Sakurai

c. As a control, the apo enzyme is completely lacking in the function. Therefore, we can undoubtedly conclude that the site which initially accepts an electron is the type 1 copper.

pH dependence of native ascorbate oxidase and its type 2 copper-depleted derivatives is of interest. As shown in Figure 3, the reaction rate of the native enzyme and type 2 copper-depleted derivatives remarkably increased with decreasing pH. The extent of the increases was more prominent than that of lactase [12] and derivatives (unpublished result). As for lactase, we supposed that the pH dependency is concerned with the increase of the redox potential of type 1 copper with decreasing pH [6d, 241. On the other hand, the redox potential of the central iron in q&chrome c is practically independent of pH over pH range 4 to 9 [22]. Although no data for the pH dependency of the redox potential of type 1 copper in ascorbate oxidase is available, the thermodynamic driving force of the reaction is expected to increase slightly as pH decreases. Otherwise, type 1 copper might become accessible more easily to the exterior of the protein through a deformation loaded on the protein structure. While we did not observe such a change on the absorption and ESR spectra, ascorbate oxidase and its type 2 copper-depleted derivatives become very unstable at below pH 5 [9d]. This will support the idea that the protein structure is easily deformed to a certain degree at a low pH and the type 1 copper site becomes more accessible to solvent or cytochrome c.

Williams et al. [161 showed that hexametaphosphate binds close to lysine-13, -86, and -87, a basic path in cytochrome c surface. Accordingly, the polyanion was expected to assist the access of both proteins by neutralizing the posi- tive charges. However, the rate of the electron-transfer became slower in the presence of an increasing amount of hexametaphosphate (Fig. 4), showing again that the association of both proteins is highly directional and the basic patch on the cytochrome c surface is not involved in the protein-protein interaction.

This study was suppotted by a Grant-in-aid for Scientific Research on Priority Areas from the A4inist~~ of Education, Science, and Culture of Japan (04225211).

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Received June 15, 1993; accepted October 26, 1993