The reactions of iso-cytochrome c2 in the photosynthetic electron transfer chain of Rhodobacter sphaeroides

Sangjin Hong#, Tim Donohue* and Antony Crofts#

#Program in Biophysics and Computational Biology, University of Illinois at Urbana- Champaign, and *Department of Microbiology, University of Wisconsin, Madison

Introduction

When the gene encoding cytochrome c2 is deleted from Rb. sphaeroides, the bacterium is unable to grow photosynthetically (1). However, spontaneous mutants appear which recover photosynthetic growth (spd mutants) (Figure 1) (1-5). Previous work has shown that these strains contain a new cytochrome, identified as a soluble cytochrome c closely related to the normal cyt c2, and called iso-cyt c2. Previous work from this collaboration has shown that the kinetics of flash-induced oxidation of c-type cytochrome in these revertant strains was slower than in wild-type strains (5). Nevertheless, the mutant strains grow rapidly under photosynthetic conditions. More recent work by Donohue and colleagues has established that deletion of the genes for both cyt c2 and iso-cyt c2 results in a strain unable to grow photosynthetically (1). There is no evidence for a membrane bound c-type cytochrome which can supplement the electron transfer function of the soluble c-type cytochromes, as is found in Rhodobacter capsulatus (6).

In this work we have used a strain of Rb. sphaeroides provided by Dr. T. Donohue in which the deletion strain was transformed with the gene for iso-cyt c2 (cycI) in plasmid, and expressed under control of an efficient promoter (Table 1). We have measured the kinetics of cytochrome changes following flash illumination under a variety of conditions to assay the behavior of the iso-cyt c2 as an electron carrier in the photosynthetic chain.

Results and Discussion

Figs. 2, 3 and 4 show the kinetics of turn-over of the reaction center (Bchl)2 (P870, measured at 542 nm), the total cytochrome c (cyt ct) measured from the difference kinetics at 551 and 542 nm (close to the maximual absorbance in the alpha-band region for cyt c2 (lmax 550 nm), iso-cyt c 2 (lmax 552 nm), and cyt c1 of the bc-complex (lmax 552 nm)), and cyt bH mesured at 561-569 nm in cells (Figure 2) and chromatophores (Figs 3 and 4) poised at different Eh ~100 mV (quinone pool 30% reduced) or at Eh ~200 mV (quinone pool oxidized), in the presence or absence of inhibitors. Apart from the slowed rate of cyt c oxidation previously observed, the kinetics are similar to those observed in wild-type strains. Since the rate-determining step in electron transfer is at the QO-site of the bc-complex, it is clear that although the rate of iso-cyt c 2 oxidation is slower in the mutant strain, it is still an order of magnitude more rapid than the rate-determining step, thus accounting for the rapid growth of these strains.

The amplitudes of the absorbance changes measured in the presence of antimycin can be used to assay the concentrations of the reaction center, bc-complex, and total cyt c, and by subtraction, the contributions of cyt c1 and iso-cyt c2 to the latter (Table 2).

The kinetics of cyt c oxidation were measured on a more rapid time in an attempt to separate contributions from the cyt c1 and iso-cyt c2. Unfortunately, the spectra are almost indetical in the alpha-band region, so the distinction must be made on the basis of kinetics. Figure 5 shows traces measured at 551-542 nm under a variety of conditions. All traces show a polyphasic kinetics, with a small fast phase (t½ < 50ms) a larger slow phase (t½ ~400ms) in the oxidation kinetics. The spectra of the two contributions are similar, with the more rapid phase showing a peak at 552.5 nm, and the slower phase at 552 nm (Figure 6). Because of the relatively small contribution of the rapid phase, we have not been able to resolve the kinetics further.

It seems likely that the rapid phase is due to oxidation of iso-cyt c2, and the slower phase to cyt c1. The relatively small amplitude of the rapid phase is consistent with the low concentration assayed from the traces of Figs. 2, 3 and 4. If this is the case, the oxidation of cyt c1 must occur through a cycling of iso-cyt c2 between the bc-complex and the reaction center, accounting for the relatively slow kinetics compared to those observed in wild-type.

References

1. Rott, M. A., Witthuhn, V. H., Schilke, B. A., Soranno, M., Ali, A., and Donohue, T. J. (1993) J. Bacteriol. 175, 358-366.

2. Rott, M. A., Fitch, J., Meyer, T. E., and Donohue, T. J. (1992) Arch. Biochem. Biophys. 292, 576-582.

3. Rott, M. A., and Donohue, T. J. (1990) J. Bacteriol. 172, 1954-1961.

4. Brandner, J. P., McEwan, A. G., Kaplan, S., and Donohue, T. J. (1989) J. Bacteriol. 171, 360- 368.

5. Donohue, T. J., McEwan, A. G., Van Doren, S., Crofts, A. R., and Kaplan, S. (1988) Biochem. 27, 1918-1925.

6. Jenny, Jr., F. E. and Daldal, F. (1993) EMBO J. 12, 1283-1292.

FIGURES & TABLES