/. Biochem. 84, 539-546 (1978)

Diffusion-Potential-Induced Oxidation and Reduction of Cytochromes in Chromatophores from Rhodopseudomonas sphaeroides

Katsumi MATSUURA and Mitsuo NTSHIMURA Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, March 27, 1978

A membrane potential jump was induced by the addition of valinomycin in the presence of a KC1 concentration gradient across the membrane of Rhodopseudomonas sphaeroides chromatophores. As well as a carotenoid band shift, which is known to be an indicator of membrane potential, absorbance changes due to the oxidation-reduction reactions of cytochromes accompanied the jump. Under aerobic conditions with no reductant added, a part of cytochrome c, was reduced by an inside-positive potential jump of about 100 mV in the time range of tens of seconds. This can be explained by the location of the cytochrome on the inner side of the chromatophore membrane and electrophoretic flow of electrons across the membrane. On the other hand, in the presence of 1 mM ascorbate, a similar jump of membrane potential induced a rapid oxidation of cytochrome c, and a subsequent reduction. A rapid reduction of 6-type cytochrome was also observed. Antimycin A inhibited the cs oxidation, but did not inhibit the b reduction. The oxidation of cytochrome cs may be explained by a diffusion-potential-induced electron flow to cytochrome b and a simultaneous electron donation by cytochrome b and cytochrome cs to a common electron acceptor, possibly a quinone.

Photosynthetic electron flow in photosynthetic bacteria begins with the release of an electron from excited bacteriochlorophyll in the reaction center complex. The flow generates a transmembrane electrochemical potential difference of protons {14). Mitchell suggested that protons were pumped by vectorial translocations of electrons across the membrane and of hydrogen atoms in the opposite direction (5). The vectorial nature of the electron transfer arm, in which the site of the primary photochemical charge separation is located, has been studied in terms of the rapid kinetics of the carotenoid band shift (2, 4) which indicates the Vol. 84, No. 3, 1978

membrane potential change (6), the effect of diffusion potential on the delayed fluorescence (7), analysis of the location of cytochrome c, which reduces photooxidized bacteriochlorophyll (8, 9), and the rapid proton binding on the outside of the chromatophore membrane after flash excitation (3). The vectonal pathway of electrons predicts the effect of membrane potential on the redox equilibrium of the constituting components. This effect was demonstrated recently by Takamiya and Dutton {10) on the equilibrium of cytochrome c, and reaction center bacteriochlorophyll in chromatophores of R. sphaeroides in the high-mem539

K. MATSUURA and M. NISHIMURA

540

brane-potential state after multiple flashes. In mitochondria, Hinkle and Mitchell (11) showed a similar poising of the redox equilibrium between cytochromes c and a by a K+ diffusion potential. Chromatophores from R. sphaeroides grown photosynthetically contain four functionally differentiated types of cytochromes, i.e., cytochromes c,, b-K, bt0, and 6m (72). Cytochrome c, has an a-band at 551 run and ZMype cytochromes at about 560 run. Illumination induces the oxidation of cytochrome c, and the reduction of fc-type cytochrome (13). In Rhodospirillum rubrum chromatophores Baltscheffsky (14) demonstrated a reduction of ft-type cytochrome and an oxidation of c-type cytochrome upon addition of PPi or ATP. This was explained in terms of reversed electron flow through a crossover point between those cytochromes. In this study, a membrane potential jump in the chromatophores from R. sphaeroides was induced by the diffusion of K+. The carotenoid band shift was monitored as an indicator of membrane potential (6, 15) and the redox changes of cytochromes c, and b were followed in terms of the absorbance changes in the a-band region. MATERIALS AND METHODS Rhodopseudomonas sphaeroides cells were grown anaerobically in the light and chromatophores were prepared as described previously (16). The preparation medium was 5 min EDTA-5 mM sodium phosphate (pH 7.4). The washed chromatophores were suspended in a small volume of 5 mM sodium phosphate (pH 7.4). 1 mM KC1 was also present throughout the preparation procedure where indicated. Membrane potential jump was induced by the addition of 130 nM valinomycin after dilution of chromatophores into a solution containing 5 mM sodium phosphate (pH 7.4) and KC1 at a given concentration. In the experiments where 1 mM sodium ascorbate was present, 30 mM sodium ascorbate was added in the concentrated chromatophore suspension (at 0°C) several hours before dilution. The membrane potential change was measured in terms of the carotenoid band shift calibrated by the K+ diffusion potential (6). Changes of cytochrome c,, cytochrome b, reaction center bacteriochlorophyll, and carotenoid

absorption were measured with a dual-wavelength spectrophotometer (Hitachi 356) as described previously (15) except for the use of a blue guard filter, Corning 9788. Spectral changes of cytochromes in the a-band region were measured with a split-beam spectrophotometer (Union Giken SM-401) connected to a data processor. RESULTS Changes at High Redox Potentials—Under aerobic conditions without reductant, only about a tenth of cytochrome c, in chromatophores was in the reduced state, and its redox potential was estimated to be about 350 mV, using a mid-point potential of 295 mV (12). Figure 1 shows the time courses of absorbance changes induced by illumination and by transmembrane potential jump at the high redox potential. A decrease in 605minus-575 nm difference absorbance indicates oxidation of the bacteriochlorophyll dimer in the reaction center (17); an increase in 562-minus-575 nm absorbance indicates reduction of b-type cytochrome; an increase in 552-minus-575 nm absorbance indicates reduction of cytochrome c,; and an increase in 523-m«uis-5O7 nm absorbance corresponds to a red shift of the carotenoid band, which is an indicator of membrane potential. The membrane potential jump in Fig. 1 induced by adding valinomycin was estimated to be 95 mV inside-positive by calibration of the carotenoid shift. 4^s5,_575 was increased by the addition of valinomycin, followed by a slower increase, corresponding mostly to the reduction of cytochrome c,. A part of the initial increase included absorbance changes other than that of cytochrome c,, in particular, the carotenoid shift. At 5 s after the potential jump, the spectrum of the change showed a rather small peak of reduced cytochrome c, at 551 nm (Fig. 2). The subsequent change had a spectrum with a clear peak of cytochrome c, reduction. The effect of carotenoid shift in the J/4M,_S76 was estimated to amount to 4% of ^su-507 from the data of Figs. 1 and 2. This value was used for correcting the difference absorbance 4/4Mf_67t in Figs. 3 and 6. The increase in /MMi-575 on illumination before the jump also seems to reflect the carotenoid shift. After the membrane potential jump, a light-induced oxidation of cytochrome c,, which had been reduced by

/. Biochcm.

DIFFUSION-POTENTIAL-INDUCED REDOX REACTIONS OF CYTOCHROMES A A

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4on toff

iValinomych

Ion toff

605-575

30 s

Fig. 1. Absorbance changes induced by light and membrane potential jump in chromatophores under aerobic conditions. The difference absorbances GO5-minus-515 nra, 562minus-575 nm, 552-minus-515 am represent the redox states of reaction-center bacteriochlorophyll, 6-typc cytochrome, and cytochrome ct, respectively, and that at 523-minus-5O7 nm, carotenoid absorbance change. The reaction mixture contained chromatophores (equivalent to 20 //M bactenochlorophyll) prepared in K+-free medium, 100 nun KC1 and 5 mM sodium phosphate, pH 7.4. 130 nM valinomycin was added at the point indicated by the arrow. The membrane potential jump upon addition was about 95 mV inside-positive, calibrated by the carotenoid change.

do-3)

-1

560 Wavelength

580

Fig. 2. Absorption spectrum changes upon membrane potential jump in the a-band region of cytochromes. The measuring beam scanned from 540 nm to 580 nm at a speed of 10 nm/s before and after valinomycin addition. The scanning started 4 s and 60 s after valinomycin addition and took 4 s to cover the wavelength range. The averages of two measurements of 4-s-after-m/raty-before (triangles) and 60-s-after-m/nttr-before (circles) were plotted. The conditions were similar to those of Fig. 1. Bactenochlorophyll concentration was 25 fiM. The dashed line represents the light-induced change before valinomycin addition. Vol. 84, No. 3, 1978

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the jump, was observed (Fig. 1). A rapid reduction of 6-type cytochrome also took place upon the addition of valinomycin. The difference spectra showed a shoulder at 663 nm. The reactioncenter bacteriochlorophyll was reduced slightly by the jump; the time course was similar to that of the reduction of cytochrome cs. Figure 3 shows the changes of cytochromes ca and b as functions of the membrane potential change. When inside-negative potential was applied, cytochrome ct changed little. With inside-positive potentials higher than about 30 mV the slope became large. The potential at which the slope changed differed somewhat from preparation to preparation, probably because of a small difference in the initial redox potential of cytochrome c,. The relationship between the changes of cytochrome b and membrane potential was linear, with a change in sign at about zero membrane potential change. Changes in the Presence of 1 mM Ascorbate—

Figure 4 shows traces of absorbance changes under conditions similar to those in Fig. 1 except for the presence of 1 mM ascorbate. Under these conditions about four-fifths of cytochrome c2 was in the reduced state. Its redox potential was calculated to be about 250 mV. However, Eh of Z>-type cytochrome was lower, because an addition

toff

of 10 /JM phenazine methosulfate, which acts as a redox mediator, reduced most of the ferricytodO"3) 3 -

<
-type cytochrome is consistent with the membrane-potential-jump-induced oxidation of cytochrome c, accompanying the reduction of b-type cytochrome. Three species of Z>-type cytochrome are known in the chromatophore of photosynthetically grown R. sphaeroides. Their mid-point potentials at pH 7.0 and a-band absorbance maxima are —90 mV, 50 mV, 155 mV and at 564-565 nm, 560 nm, 558559 nm, respectively (12). The difference spectrum induced by the inside-positive potential jump in

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the presence of 1 mM ascorbate (Fig. 5) had a broad peak from 560 to 565 nm. This may indicate that more than a single species of b-type cytochrome is located on the inner side of the membrane and that the redox component on the outer side of the site-II electrogenic pathway is not 6-type cytochrome. It was not determined which of the cytochrome b species caused the oxidation of cytochrome c,. However, the above argument for two parallel pathways of electron transfer across the membrane, in which cytochrome c, and cytochrome b linked to a common carrier are involved, can be applied to each species with little modification.

REFERENCES 1. Jackson, J.B., Crofts, A.R., & von Stedingk, L.-V. (1968) Eur. J. Biochem. 6, 41-54 2. Jackson, J.B. & Crofts, A.R. (1971) Eur. J. Biochem. 18, 120-130 3. Cogdell, R.J., Jackson, J.B., & Crofts, A.R. (1973) /. Bioenergetics 4, 211-227 4. Jackson, J.B. & Dutton, P.L. (1973) Blochim. Biophys. Acta 325, 102-113 5. Mitchell, P. (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation Glynn Research, Bodmin, Cornwall 6. Jackson, J.B. & Crofts, A.R. (1969) FEBS Lett. 4, 185-189 7. Fleischman, D E. (1971) Photochem. Photobiol. 14, 227-286

8. Prince, R C, Baccarini-Melandri, A., Hauska, G.A., Melandri, B.A., & Crofts, A.R. (1975) Biochim. Biophys. Ada 387, 212-227 9. Hochman, A., Fndberg, I., & Carmeli, C. (1975) Eur. J. Biochem. 58, 65-72 10. Takamiya, K. & Dutton, P.L. (1977) FEBS Lett. 80, 279-284 11. Hinkle, P. & Mitchell, P. (1970) /. Bioenergetics 1, 45-60 12. Dutton, P.L. & Jackson, J.B. (1972) Eur. J. Biochem. 30, 495-510 13. Nishimura, M. & Chance, B. (1963) Biochim. Biophys. Acta 66, 1-16 14. Baltscheffsky, M. (1967) Biochem. Biophys. Res. Commun. 28, 270-276 15. Matsuura, K. & Nishimura, M. (1977) Biochim. Biophys. Acta 459, 483-491 16. Matsuura, K. & Nishimura, M. (1977) Biochim. Biophys. Acta 462, 700-705 17. Dutton, P.L., Petty, K.M., Bonner, H.S., & Morse, S.D. (1975) Biochim. Biophys. Ada 387, 536-556 18. Hochman, A. & Carmeli, C. (1977) Arch. Biochem. Biophys. 179, 349-359 19. Prince, R.C. & Dutton, P.L. (1977) Biochim. Biophys. Acta 462, 713-747 20. Baltscheffsky, M. (1975) in Proceedings of the 3rd International Congress on Photosynthesis (Avron, M., ed.) pp. 799-806, North Holland Publishing Co., Amsterdam 21. Mitchell, P. (1976) J. Theor. Biol. 62, 327-367 22. Erecinska, M., Chance, B., Wilson, D.F., & Dutton, P.L. (1972) Proc. Natl. Acad. Sci. U.S. 69, 50-54 23. Erecinska, M. & Wilson, D.F. (1972) FEBS Lett. 24, 269-272

/. Biochem.

Diffusion-potential-induced oxidation and reduction of cytochromes in chromatophores from Rhodopseudomonas sphaeroides.

/. Biochem. 84, 539-546 (1978) Diffusion-Potential-Induced Oxidation and Reduction of Cytochromes in Chromatophores from Rhodopseudomonas sphaeroides...
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