Mechanism of Ascorbic Acid Oxidation by Cytochrome b561

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11910 Biochemistry, Vol. 40, No. 39, 2001 Njus et al. of initial rates limits the data to reduction of the first heme. Rapid electron transfer between two hemes would not affect the quantitation of reduction, because the hemes have indistinguishable absorption spectra (19). According to the mechanism described above, the bound ascorbate monoanion acts as an electron donor and forms the bound semidehydroascorbate radical as an intermediate. The monoanion in solution is a relatively poor electron donor having a reduction potential of 0.766 V. If it is to reduce cytochrome b 561 (E° ) 0.140 V), then the ascorbate-binding site must destabilize the monoanion and stabilize the free radical to facilitate the electron transfer reaction. Molecular modeling shows that hydrogen bonding with the imidazole may accomplish this if the unprotonated imidazole is hydrogen bonded to the 2-hydroxyl of the ascorbate monoanion. The hydrogen bond energy at this location is relatively stronger for the free radical than for the monoanion. Hydrogen bond energies at the other two oxygen atoms are stronger for the monoanion than for the free radical. Furthermore, minimized structures of imidazole-ascorbate complexes hydrogen bonded through C 2 show that the various forms of ascorbate (monoanion, free radical, and radical anion) all have similar atomic coordinates. Thus, reorientation of the ascorbate-histidine complex does not need to occur as the cytochrome proceeds through the reduction reaction. The bound ascorbate free radical must dissociate from the site and lose a proton to form the semidehydroascorbate radical anion. This could happen by (1) the bound radical dissociating and then deprotonating, (2) the bound radical losing a proton and then dissociating as the semidehydroascorbate radical anion, or (3) the bound radical transferring a proton to the binding site and then dissociating as semidehydroascorbate. The third case is consistent with the kinetics of oxidation by semidehydroascorbate (Figure 4). The pH dependence of the rate constant suggests that the semidehydroascorbate radical anion reacts with a protonated site having a pK of ∼6.5. The other two possibilities are energetically improbable. In the first case, stabilization of the bound radical sufficient to achieve a reasonable reduction potential for electron donation (0.3 V) would require an extremely tight binding affinity (K D ) 10 -11 M). In the second case, either the semidehydroascorbate would have to bind with very high affinity or the pK of the bound radical would have to increase substantially. To achieve a dissociation constant of 1 µM with a reasonable reduction potential, the pK would have to increase to 5 from the value of -0.45 observed in solution. The third case, by contrast, can be achieved with reasonable energetics. Thermodynamic equilibrium in this system (Figure 7) relates the parameters K Dm , E° c , K Dr ,pK r ,pK c , and E° f : E° c ) E° f + (RT/F log e)(log K Dr - log K Dm - pK c + pK r ) (5) FIGURE 8: Dependence of the reduction potential of bound ascorbate on the dissociation constant for the semidehydroascorbate radical anion. Lines were calculated using eq 5 with the following values: K Dm ) 1 mM, E° f ) 0.766 V, pK r )-0.45, and T ) 295 K. Values used for pK c are 6.0 (top line), 6.5 (middle line), and 7.0 (bottom line). Knowing E° f ) 0.766 V, K Dm ) 1 mM, and pK r )-0.45, we can determine E° c as a function of K Dr and pK c (Figure 8). pK c , the pK of the active site residue, is ∼6 or∼7. K Dr , the binding constant for semidehydroascorbate, is unknown, but in studies of cytochrome b 561 oxidation, we could not observe saturation by semidehydroascorbate up to concentrations of 1 µM (18). Consequently, K Dr is expected to be greater than 10 -6 M. Semidehydroascorbate oxidizes cytochrome b 561 10 3 times faster than ascorbate reduces it (18). This is consistent with the midpoint potential of the bound ascorbate or semidehydroascorbate being ∼300 mV, 160 mV more positive than the midpoint potential of the cytochrome (140 mV; 17, 19). A midpoint potential of this magnitude would be compatible with a dissociation constant for semidehydroascorbate (K Dr ) of ∼100 µM (Figure 8). These values not only are reasonable but also show that the mechanism outlined in Figure 7 does not involve any steps requiring large changes in free energy. The reactions, therefore, are all readily reversible, a necessity for a cytochrome whose function is to equilibrate ascorbate and semidehydroascorbate. The mechanism proposed here hypothesizes that the reactivity of ascorbate is controlled by the ascorbate-binding site. The heme serves only as an acceptor in the electron transfer reaction. As a consequence, ascorbate bound to cytochrome b 561 is poised for electron donation to any suitable electron acceptor. If the heme is already reduced, then the bound ascorbate may react adventitiously with another oxidant. Of particular interest is molecular oxygen, which may be reduced to the superoxide radical anion, initiating the production of a variety of damaging oxygen •- radicals. The reduction potential of the O 2 /O 2 pair is ∼300 mV under physiological conditions (1, 26). To avoid generation of additional superoxide, it is obviously advantageous for the bound ascorbate to have a midpoint potential that is g300 mV, implying a binding constant for semidehydroascorbate that is 100 µM or weaker (Figure 8). As discussed above, these are likely values. According to the Marcus theory for electron transfer reactions in solution (27), outer-sphere electron transfer reactions are spontaneous and occur at rates that depend solely on the intrinsic reactivity of the compounds involved and on the difference in their relative reduction potentials. Such reactions are obviously not well suited to biological systems in which reaction rates are carefully regulated and desired redox reactions often involve reactants with relatively
Mechanism of Ascorbic Acid Oxidation by Cytochrome b 561 Biochemistry, Vol. 40, No. 39, 2001 11911 close reduction potentials as, for example, in respiratory and other redox chains. The mechanism presented here suggests that biological systems have brought redox reactions under control by selecting redox compounds that do not react by outer-sphere electron transfer but can be made to behave as electron donors and acceptors when bound in an appropriate site. The function of the redox enzyme, therefore, is to provide both a site for unlocking the electron-transferring capability of the substrate and also a redox center with which the bound substrate can react, preempting spontaneous reactions with other undesired reactants. An attraction of this perspective is that, like the Marcus theory for uncatalyzed reactions, it allows enzymatic redox reactions to be analyzed, at least to a first approximation, in terms of the separate properties of the reactants: a complexed organic substrate and the enzyme’s metal center. REFERENCES 1. Njus, D., and Kelley, P. M. (1991) FEBS Lett. 284, 147- 151. 2. Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30. 3. Srivastava, M., Duong, L. T., and Fleming, P. J. (1984) J. Biol. Chem. 259, 8072-8075. 4. Wakefield, L. M., Cass, A. E. G., and Radda, G. K. (1986) J. Biol. Chem. 261, 9739-9745. 5. Menniti, F. S., Knoth, J., and Diliberto, E. J., Jr. (1986) J. Biol. Chem. 261, 16901-16908. 6. Njus, D., and Kelley, P. M. (1993) Biochim. Biophys. Acta 1144, 235-248. 7. Smith, A. D., and Winkler, H. (1967) Biochem. J. 103, 480- 482. 8. Njus, D., and Radda, G. K. (1979) Biochem. J. 180, 579- 585. 9. Silsand, T., and Flatmark, T. (1974) Biochim. Biophys. Acta 395, 257-266. 10. Duong, L. T., Fleming, P. J., and Russell, J. T. (1984) J. Biol. Chem. 259, 4885-4889. 11. Wakefield, L. M., Cass, A. E. G., and Radda, G. K. (1984) J. Biochem. Biophys. Methods 9, 331-341. 12. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P. M. W., Johnson, B. G., Robb, M. A., Cheeseman, J. R., Keith, T., Petersson, G. A., Montgomery, J. A., Raghavachari, K., Al- Laham, M. A., Zakrzewski, V. G., Ortiz, J. V., Foresman, J. B., Cioslowski, J., Stefanov, B. B., Nanayakkara, A., Challacombe, M., Peng, C. Y., Ayala, P. Y., Chen, W., Wong, M. W., Andres, J. L., Replogle, E. S., Gomperts, R., Martin, R. L., Fox, D. J., Binkley, J. S., Defrees, D. J., Baker, J., Stewart, J. P., Head-Gordon, M., Gonzalez, C., and Pople, J. A. (1995) GAUSSIAN 94, Gaussian, Inc., Pittsburgh, PA. 13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85. 14. Tirrell, J. G., and Westhead, E. W. (1979) Neuroscience 4, 181-186. 15. Jalukar, V., Kelley, P. M., and Njus, D. (1991) J. Biol. Chem. 266, 6878-6882. 16. Kipp, B. H., Kelley, P. M., and Njus, D. (2001) Biochemistry 40, 3931-3937. 17. Flatmark, T., and Terland, O. (1971) Biochim. Biophys. Acta 253, 487-491. 18. Kelley, P. M., Jalukar, V., and Njus, D. (1990) J. Biol. Chem. 265, 19409-19413. 19. Apps, D. K., Boisclair, M. D., Gavine, F. S., and Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 8-16. 20. Takeuchi, F., Kobayashi, K., Tagawa, S., and Tsubaki, M. (2001) Biochemistry 40, 4067-4076. 21. Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F., and Tagawa, S. (2000) Biochemistry 39, 3276-3284. 22. Degli Esposti, M., Kamenskiy, Y. A., Arutjunjan, A. M., and Konstantinov, A. A. (1989) FEBS Lett. 254, 74-78. 23. Burbaev, D. S., Moroz, I. A., Kamenskiy, Y. A., and Konstantinov, A. A. (1991) FEBS Lett. 283, 97-99. 24. Tsubaki, M., Nakayama, M., Okuyama, E., Ichikawa, Y., and Hori, H. (1997) J. Biol. Chem. 272, 23206-23210. 25. Kamensky, Y. A., and Palmer, G. (2001) FEBS Lett. 491, 119-122. 26. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. ReV. 59, 527-605. 27. Marcus, R. A., and Sutin, N. (1985) Biochim. Biophys. Acta 811, 265-322. BI010403R
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Mechanism of Ascorbic Acid Oxidation by Cytochrome b561

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