INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

In neurosecretory vesicles, such as adrenal chromaffin vesicles and pituitary neuropeptide secretory vesicles, intravesicular ascorbate (AsA )¹ functions as the electron donor for copper-containing monooxygenases such as dopamine -monooxygenase and peptidyl-glycine -amidating monooxygenase (1). Upon these monooxygenase reactions, monodehydroascorbate (MDA) radical is produced by univalent oxidation of AsA (2, 3). It is believed that the intravesicular MDA radical is reduced back to AsA by membrane-bound cytochrome b₅₆₁, and subsequently cytochrome b₅₆₁ is reduced by extravesicular AsA (4-7). Thus, cytochrome b₅₆₁ is likely to serve as an electron shuttle, maintaining the AsA concentration inside the vesicles.

Cytochrome b₅₆₁ is a highly hydrophobic hemoprotein with a molecular mass of ~28 kDa and contains five or six transmembrane -helices (8, 9). It had been widely accepted that cytochrome b₅₆₁ contains only one b-type heme per molecule, by analyses with pyridine hemochrome and Western blotting methods for quantitation of heme and apoprotein, respectively (10-13). However, very recently, we have established a new purification procedure of cytochrome b₅₆₁ from bovine adrenal chromaffin vesicles (14). We found that the purified cytochrome b₅₆₁ contained two b-type hemes per molecule. In addition, each heme b center exhibited independent EPR signals in the oxidized state. Based on these results and comparison of the amino acid sequences of cytochrome b₅₆₁ from various species, we have proposed that the two heme prosthetic groups are located on both sides of the membrane in close contact with AsA and MDA binding sites, respectively, to facilitate the electron transfer across the membranes (14, 15). However, the role of each of the two heme b centers has not been elucidated.

Because of the instability of the MDA radical, which disproportionates rapidly to dehydroascorbate and AsA , the electron transfer reaction with cytochrome b₅₆₁ was measured indirectly in the presence of AsA and ascorbate oxidase (5, 6). However, it is possible to investigate directly the reaction of MDA radical using a pulse radiolysis technique (16-20). We could observe electron donations to MDA radical from hepatic NADH-cytochrome b₅ reductase (19) and from MDA reductase purified from cucumber chloroplasts (20). In particular, MDA reductase was shown to be a good electron donor for MDA radical (20). The present study describes a successful application of the pulse radiolysis technique to investigate the reaction of MDA radical with cytochrome b₅₆₁ purified from bovine adrenal chromaffin vesicles. We obtained clear evidence that the two heme b centers in cytochrome b₅₆₁ have distinct roles in the reaction with MDA radical and AsA , respectively.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Purification of Cytochrome b₅₆₁-- Cytochrome b₅₆₁ was purified to a homogeneous state, as reported previously (14). The purity of cytochrome b₅₆₁ was analyzed with visible absorption spectra and SDS-polyacrylamide gel electrophoresis. Before use, cytochrome b₅₆₁ was passed through a Sephadex G-25 column equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 1.0% (w/v) -octyl glucoside and 1.0 mM AsA . The passed fractions of cytochrome b₅₆₁ were concentrated in the Amicon concentrator. All other reagents were commercially obtained as the analytical grade. The concentration of cytochrome b₅₆₁ was determined using a millimolar extinction coefficient of 267.9 mM¹ cm¹ at 427 nm in the reduced state (14).

Pulse Radiolysis-- Samples of cytochrome b₅₆₁ for pulse radiolysis were prepared as follows. Solutions containing 10 mM potassium phosphate buffer (pH 5-8), 1% -octyl glucoside, and 5 mM AsA were bubbled with N₂O gas for 5 min. Then, a concentrated solution of cytochrome b₅₆₁ was added to the solution to make an appropriate final concentration as indicated in the figure legends.

1

1

Treatment in Alkaline pH in the Oxidized Form-- Purified cytochrome b₅₆₁ (~100 µM) in 10 mM potassium phosphate buffer (pH 7.0 and 8.4) containing 1.0% -octyl glucoside was oxidized with stepwise additions of potassium ferricyanide (100 mM) solution. The fully oxidized cytochrome b₅₆₁ samples were kept in the dark overnight on ice. The samples thus obtained were passed through Sephadex G-25 equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 1.0% (w/v) -octyl glucoside and 1.0 mM AsA . Optical absorption spectra were recorded with a UVIKON 922 (Kontron), a UV-2200 A (Shimadzu), or a Hitachi U-3000.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

A transient spectrum of MDA radical with an absorbance maximum at 360 nm was observed 100 ns after pulse radiolysis of N₂O-saturated aqueous solutions in the presence of 5 mM AsA and 1.0% -octyl glucoside. Under the conditions employed here, the primary species (hydrated electron (e_aq), OH^·, H^·) generated by pulse radiolysis of aqueous solutions were efficiently converted to MDA radical at an approximate concentration of 20-30 µM (17-20). The MDA radicals thus formed reacted rapidly with the reduced form of cytochrome b₅₆₁. A decrease at 430 nm and an increase at 405 nm reflected this reaction (Fig. 1A). The kinetic difference spectrum obtained 20 ms after the pulse is similar to that of the difference spectrum of the oxidized minus reduced forms of cytochrome b₅₆₁ (Fig. 1B). It is therefore concluded that MDA radical reacts with the reduced form of cytochrome b₅₆₁ to produce the oxidized form, as shown in Reaction 1.  For determination of the rate constant of Reaction 1, the concentration of MDA radical was lowered to 1-2 µM MDA radical, and the cytochrome b₅₆₁ concentration was varied between 46 and 100 µM. Fig. 2 shows the concentration dependence of cytochrome b₅₆₁ on the apparent rate constants. From the slope of Fig. 2, the second-order rate constant of the reaction was calculated to be 2.6 × 10⁶ M¹ s¹ at pH 7.0. This value is in good agreement with the one reported previously (1.2 × 10⁶ M¹ s¹ at pH 7.0) in which a steady-state kinetics method was used employing chromaffin vesicle membranes and an AsA -ascorbate oxidase system (7).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Absorbance changes after pulse radiolysis of the reduced form of cytochrome b561 measured at 430 and 405 nm in the presence of AsA and N2O at pH 7.0 (A), and comparison of the kinetic difference spectrum () for cytochrome b561 at 20 ms after pulse radiolysis with the reduced minus oxidized difference spectrum (- - -) of cytochrome b561 (B). The reaction mixture contained 5.2 µM cytochrome b561, 5 mM AsA , 1% -octyl glucoside, 10 mM potassium phosphate buffer at pH 7.0. Note that the left ordinate is in units of extinction coefficients corresponding to the reduced minus oxidized spectrum, while the right ordinate is in units of A corresponding to the kinetic difference spectrum.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of the apparent rate constants of the reaction of the reduced form of cytochrome b561 with MDA radical. The rates were determined by the absorption change at 561 nm. The reaction medium contained 5 mM AsA , 1% -octyl glucoside, 10 mM potassium phosphate buffer at pH 7.0, and 1-2 µM MDA radical.

Subsequently, the initial changes in absorbance reversed in the time range of seconds (Fig. 3), indicating that re-reduction of cytochrome b₅₆₁ occurred. The rate constant of this process increased with increases in the concentration of AsA (data not shown). This indicates that the reduction process is a consequence of a bimolecular reaction of AsA with the oxidized form of cytochrome b₅₆₁, as shown in Reaction 2.  The second-order rate constant of the reaction was calculated to be 8 × 10² M¹ s¹ at pH 7.0. The value is also good agreement with the value reported previously (4.5 × 10² M¹ s¹) in which a stopped-flow method was employed to analyze the electron transfer reaction from AsA to oxidized cytochrome b₅₆₁ in chromaffin vesicle membranes (7, 24).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Absorbance changes after pulse radiolysis of the reduced form of cytochrome b561 measured at 430 and 405 nm in the presence of AsA and N2O at pH 7.0. Experimental conditions were the same as in Fig. 1.

The effects of pH on the oxidation rates of cytochrome b₅₆₁ with MDA radical and for reduction with AsA were examined. It is evident that pH profiles for the oxidation and the reduction reactions are different, as shown in Fig. 4. The rate constant for the oxidation of cytochrome b₅₆₁ increased with decreasing pH. The maximum reaction rate of 4.3 × 10⁶ M¹ s¹ was obtained at pH 5.5. The pH-dependent change could be fitted to a single deprotonation process with a pK_a value of 6.7. In contrast, the rate constants of the reduction of cytochrome b₅₆₁ increased with increasing pH in the range of 5 to 6.5 and then decreased with increasing pH in the range of 7 to 8. The maximum reaction rate constant of 8 × 10² M¹ s¹ was obtained around pH 6.8. 

View larger version (10K): [in this window] [in a new window]   Fig. 4.   pH dependence of the rate constants of the reaction of MDA radical with the reduced form of cytochrome b561 (A) and the reaction of AsA with the oxidized form of cytochrome b561 (B). Experimental conditions were the same as in Fig. 2.

To elucidate the contribution of two heme b centers in the electron transfer reaction, MDA radical concentration dependence was examined. Under the experimental conditions employed, the concentration of MDA radical could be varied between 2 and 20 µM by attenuating the dose of the electron beam whereas the concentration of the reduced cytochrome b₅₆₁ was maintained constant at 12 µM. As shown in Fig. 5, the oxidized cytochrome b₅₆₁ by the reaction (at 20 ms after the pulse) increased with increasing the concentration of MDA radical up to 5 µM, and it reached a plateau in the range 8 to 20 µM. A distinct inflection was observed at a point of ~0.5 eq of MDA radical to cytochrome b₅₆₁. It is noteworthy that only half of the heme center could be oxidized with an excess concentration of MDA radical.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   MDA radical concentration dependence on the oxidation of cytochrome b561 observed after pulse radiolysis. The reaction mixture contained 12 µM cytochrome b561, 5 mM AsA , 1% -octyl glucoside, 10 mM potassium phosphate buffer at pH 7.0.

In a previous report, we showed that incubation of oxidized cytochrome b₅₆₁ in a mild alkaline condition specifically depletes the electron-accepting ability from AsA for about one-half of the heme centers (15). Fig. 6A shows the visible spectra of cytochrome b₅₆₁ (pre-treated at pH 8.2 and 7.0, respectively, in the oxidized state) in the presence of AsA . Addition of 5 mM AsA to the alkaline treatment of cytochrome b₅₆₁ caused a reduction of only about half of the heme centers, whereas all the heme centers of cytochrome b₅₆₁ treated at pH 7.0 (control sample) could be reduced. Fig. 6, B and C, shows the absorbance changes at 430 nm after pulse radiolysis of these samples. The absorbance changes of the control sample after the pulse were not affected. For the alkaline-treated sample, on the other hand, the decrease in absorbance at 430 nm after the pulse was very small, although about half of the heme b centers were in the reduced state. The slower second phase (i.e. the absorbance increase at 430 nm due to the re-reduction of oxidized cytochrome b₅₆₁ with AsA ) was completely lost. These results suggest that one of the heme centers, responsible for electron donation to the MDA radical, is selectively modified upon the alkaline treatment.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of treatment at pH 7.0 (- - -) and pH 8.2 (------) on the visible spectra of cytochrome b561 (A) and absorbance changes at 430 nm after pulse radiolysis of the treated cytochrome b561 at pH 7.0 (B) and pH 8.2 (C) in the presence of AsA and N2O at pH 7.0. Alkaline-treated (at pH 8.2 for 20 h and then restored to pH 7.0) and control (incubated at pH 7.0) samples of cytochrome b561 in the oxidized state were prepared as described in the text. Solid sodium ascorbate was added (5 mM), and the spectra were recorded immediately. Other conditions were the same as in Fig. 1.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The present study clearly shows that one of the heme b centers in cytochrome b₅₆₁ specifically reacts with MDA radical, whereas the other does not. This is verified by the MDA radical concentration dependence on the oxidization of heme b after the pulse as shown in Fig. 5. Almost stoichiometric oxidation of heme b was observed under the condition of [MDA radical] < [1/2 of cytochrome b₅₆₁], whereas only one-half of heme b was oxidized at excess MDA radical concentrations. In addition, the distinct functions of two heme b centers were revealed by pulse radiolysis experiments on the alkaline-treated sample. Our previous report showed that one of the heme centers is very labile to the alkaline treatment, whereas the other heme retains the ability to accept electrons from AsA (15). It is likely that one of the two heme centers, which is very labile to the alkaline treatment, participates in the electron donation to MDA radical, because very slight oxidation of the heme center was observed after pulse radiolysis of the alkaline-treated sample.

EPR spectra of the purified cytochrome b₅₆₁ in the oxidized state showed the presence of two distinct heme b species (14). One of them shows usual low spin signals and is very similar to those of microsomal cytochrome b₅ (25), chloroplast cytochrome b₅₅₉ (26), and cytochrome b of bo-type ubiquinol oxidase (27), all of which are known to have bisimidazole ligands. The other species shows a highly anisotropic low spin signal (g_z = 3.70) with a lower redox potential and is very similar to those of cytochrome b of the mitochondrial complex III (28, 29) and chloroplast cytochrome b₆ (30). The presence of two independent heme centers was supported by the observation of two potentiometrically different forms of cytochrome b₅₆₁ determined by an optical potentiometric technique (31). We could not identify definitely which heme center is responsible for the electron donation to MDA radical, but it is very likely that the heme center with the lower redox potential (the g_z = 3.70 species) participates in the electron-accepting reaction from the extravesicular AsA . On the other hand, the usual low spin heme species (the g_z = 3.14 species) with a higher redox potential is responsible for the electron donation to MDA radical. This assumption is reasonable since the intramolecular electron transfer of cytochrome b₅₆₁ occurs from extravesicular to intravesicular to sides. This is also consistent with the result of the alkaline treatment experiment. Indeed, the heme center having the g_z = 3.14 signal can be converted to another form (the g_z = 2.84 species) upon elevation of pH, whereas the other heme center (the g_z = 3.70 species) showed only a slight pH-dependent spectral change (14).

Following the electron donation to MDA radical from the heme with a higher redox potential, an intramolecular electron transfer to the oxidized heme from the other heme with a higher redox potential should take place. The expected intramolecular electron transfer, however, could not be followed directly by the present method, because the two heme centers have indistinguishable visible spectra (14, 31). If the rate of the intramolecular electron transfer (k_i) is much faster and the rate-determining step is the oxidation of the heme center with MDA radical (k_i > k₁ [MDA radical]), the two hemes would be oxidized simultaneously. In the present work, however, only half of the heme in cytochrome b₅₆₁ was oxidized under excess MDA radical concentration. This fact indicates that the intramolecular electron transfer must occur later than 1 ms region. In our previous studies, the intramolecular electron transfer in the region of milliseconds was observed for copper-containing nitrite reductase (21, 22) and cytochrome cd₁ nitrite reductase (23). However, the two redox centers in these proteins are only 10 to 20 Å apart (32, 33). In cytochrome b₅₆₁, the two heme centers are expected to be located on both sides of vesicular membranes (15), and therefore the distance between these two redox centers might be 40 to 50 Å. Thus, an intramolecular electron transfer in the range of seconds may be quite reasonable.

The pH dependence of the rate constants of the electron transfer reactions (Fig. 4) provides further insight into the function of cytochrome b₅₆₁. The optimal pH of the oxidation (pH = 5.5) and the reduction (pH 6.8) of the heme centers of cytochrome b₅₆₁ correspond to the physiological pH at the intra- and extravesicular sides, respectively. Recently, we have proposed a plausible structural model of cytochrome b₅₆₁ on the basis of a comparison of the deduced amino acid sequences of seven species (15). In the model, there are two fully conserved regions in the sequences; the first conserved sequence (⁶⁹ALLVYRVFR⁷⁷) is located on the extravesicular side of -helical segment, and the second one (¹²⁰SLHSW¹²⁴) is located in an intravesicular loop connecting two -helical segments. Since these conserved sequences are likely to form the binding sites for extravesicular AsA and intravesicular MDA, respectively, the present results support our proposal that the two heme b centers are located on both sides of the vesicular membranes in close contact with the AsA - and MDA-binding sites (15).

It is important to note that this is the first direct observation of electron transfer between a ferrous hemoprotein and an MDA radical generated by pulse radiolysis. In the previous report, we could not observe such electron transfer reactions with MDA radical for several ferrous hemoproteins (19), although the reactions are expected to be energetically favorable on the basis of redox potential differences between MDA/AsA (E_m,7 = 330 mV) (34) and hemoproteins (35). It is evident that, for biological molecules, the occurrence of electron transfer with MDA radical cannot be predicted simply in terms of the redox potentials (19). The highest rate constant for the reaction of MDA radical with biological molecules was obtained for MDA reductase (2.6 × 10⁸ M¹ s¹) (20). We have proposed previously that the high rate constant of the enzymatic reaction might be governed by both the specific geometry of substrate within the active site and the redox potential difference. The facilitated electron transfer reaction in MDA reductase is likely to be provided by several cationic amino acid residues near the active site, which may have a role in electrostatic guidance of the anionic MDA radical substrate to the active center (20). It is noteworthy that, in the intravesicular side of cytochrome b₅₆₁, there are several conserved positively charged residues located in the loop connecting helices 3 and 4, and the fully conserved sequence (¹²⁰SLHSW¹²⁴) follows this region immediately (15).

In conclusion, the following unique properties of cytochrome b₅₆₁ have now become evident. First, an efficient transmembrane electron transfer is catalyzed by this hemoprotein, using AsA as a physiological electron supply. Second, the two heme b centers have distinct roles for the electron donation to MDA radical and the electron acceptance from AsA . Further studies are in progress to reveal the structural properties of this cytochrome.