Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid.

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.


Cytochrome
bsel transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome bssl in adrenal medullary chromaffin vesicle membranes and external ascorbatelsemidehydroascorbate were measured. The reduction of cytochrome bhel by external ascorbate may be measured by a stopped-flow method.
The rate constant is 450 (2 190) M-' s-' at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome bbB1 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (A 0.5) x lo6 M-l s-' at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b 561 and ascorbate/semidehydroascorbate.
These results suggest that cytosolic ascorbate will reduce cytochrome b&G1 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b6el shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.
In secretory vesicles, such as adrenal medullary chromaffin vesicles, ascorbic acid functions as the electron donor for at least two intravesicular monooxygenases: dopamine /3-monooxygenase and peptidyl-glycine a-amidating monooxygenase. The intravesicular store of ascorbate is maintained by cytochrome bsel, a membrane protein that imports electrons into the vesicles to reduce the semidehydroascorbate produced by the monooxygenases back to ascorbate (Njus et al., 1983(Njus et al., , 1986Wakefield et al., 1982). The likely external source of electrons is cytosolic ascorbic acid (Levine et al., 1985;Menniti et al., 1986;Beers et al., 1986;Herman et al., 1988). Thus, the cytochrome serves as an electron shuttle, maintaining ascorbate inside the vesicles at the expense of ascorbate on the outside.
The pH gradient across the chromaffin vesicle membrane creates a thermodynamic force driving this electron flow. This is because the midpoint reduction potential of ascorbic acid is pH-dependent (Iyanagi et al., 1984) being 90 mV higher at the intravesicular pH (5.5) than at the cytosolic pH (7.0). Since the midpoint reduction potential of cytochrome b,,, does not depend on pH (Apps et al., 1984), ascorbate should reduce the cytochrome more readily at higher pH and semidehydroascorbate should oxidize it more readily at lower pH. To prove that ascorbate reduces cytochrome &I quickly enough to serve as the physiological cytosolic electron donor, we have measured rates of electron transfer between cytochrome &I and external ascorbate/semidehydroascorbate using two experimental strategies. To confirm the expected pH dependence of the electron transfer reactions, we have determined these rates as a function of pH.

AND DISCUSSION
Reduction of Cytochrome b,,, by External Ascorbic Acid-The rate of reduction of cytochrome bSel by external ascorbic acid may be observed directly using the stopped-flow method (Fig. IA). In this experiment, ascorbate-free ghosts are employed so the cytochrome is obtained in a completely oxidized state. Upon mixing with ascorbate (AH-),* the cytochrome becomes reduced (Fig. 2); the initial rate of cytochrome bSel reduction is proportional to the ascorbate concentration at least at low [AH-] (Fig. 3).
The simplest kinetic analysis assumes that this reaction is characterized by a rate constant that is first-order in each substrate.
AH,, + B,, "-14 Ad;, + Bred + H' (1) Then, the rate constant kelA is equal to the initial rate divided by the ascorbate and cytochrome concentrations (Equation

4, Supplementary
Material) and is given by the slopes of the plots shown in Fig. 3. Values for k-,* determined in this way increase slightly with pH (Table I).
Although determined from initial rates of cytochrome reduction, kmlA should also account for the subsequent time course of the stopped-flow experiment (Fig. 2). Since the back reaction (Reaction II) may become important at later times, it must also be considered. The rate constant for the back reaction (I2,*) may be calculated from the measured value of LIA, because their ratio is equal to the equilibrium constant, which can be calculated from the midpoint reduction potentials of the two redox pairs. klAlk-,* = K,, = expl@% -E&W/R771 (1) Also important is the disproportionation of the ascorbate free radical which is characterized by a second-order rate constant k dw Ad+A-+H+%*H-+* (III) When the rate equations for these reactions are numerically integrated, it is apparent that a good fit is obtained at earlier times and divergence occurs only later as accumulated error becomes large (Fig. 2). This shows that the back reaction accounts at least in part for the nonexponential shape of the time course; semidehydroascorbate, generated as cytochrome bbG1 is reduced, slows the reduction rate at later times.
Oxidation of Cytochrome b,,, by External Semidehydroascorbate-The rate of oxidation of cytochrome b5G1 by externa1 semidehydroascorbate may be determined by a steady-state method (Fig. 1B). In this experiment, ascorbate-loaded ghosts are employed so the cytochrome is obtained in a mostly reduced state. When these ghosts are suspended in an ascorbate-containing medium and ascorbate oxidase is added, cytochrome bsG1 becomes transiently oxidized (Fig, 4B). Ascorbate oxidase oxidizes ascorbate to semidehydroascorbate (Yamazaki and Piette, 1961) and semidehydroascorbate in turn oxidizes cytochrome bsG1 (Kelley and Njus, 1986 one done at pH 6.0 (0) and one done at pH 8.0 (0).
The straight lines, fit to the data points by linear regression, have slopes of 4.04 X lo-' l/g. min and 11.2 X lo-* l/g.min, respectively. These slopes are measures of the rate constant km,*.  Fig. 3. Measurements of klA were taken from plots of the kind shown in Fig. 5  The semidehydroascorbate concentration may be measured under the same conditions but in the absence of ghosts (Fig.  4A). It is evident that the oxidation of the cytochrome parallels the presence of semidehydroascorbate.
The low semidehydroascorbate concentration at pH 6.0 is consistent with rapid disproportionation at low pH (Bielski et al., 1981). The data in Fig. 4A may be used to calculate the disproportionation constant of semidehydroascorbate.
Disproportionation constants determined in this way are pH-dependent and in good agreement with those given in the literature (Table II) Then, a value for kl* may be computed for any time in the steady-state experiment using Equation 9 (see Supplementary  Material). Values are reasonably time-independent until later times when accumulated error becomes large (Fig. 5).  (Iyanagi et al., 1984), the midpoint potential for cytochrome &I can be calculated. As shown in Table I, this agrees well with the measured value of +0.14 V (Flatmark and Terland, 1971;Apps et al., 1984). This consistency is strong evidence that the two methods both give accurate estimates of the rate constants.
The rate constant for the reduction of cytochrome b561 by external ascorbic acid is 4.3 x 10m2 I/g.min at pH 6.0 (Table  II). The rate constant for the reduction of cytochrome bb6, by internal ascorbic acid, measured previously (Kelley and Njus, 1988), is 6.6 x 10m3 l/g.min.
This indicates that the cytochrome reacts considerably faster with external than with internal ascorbate. Since the cytochrome is designed to transfer electrons from external ascorbate to internal semidehydroascorbate, reduction by external ascorbate may be kinetically favored over reduction by internal ascorbate.
Finally, the redox state of cytochrome bsG1 in uiuo may be considered. To maintain steady state electron flow across the membrane, external ascorbate must reduce cytochrome bsel at the same rate as the cytochrome is oxidized by internal semidehydroascorbate.