The reactions of iso-cytochrome c2 in the photosynthetic electron transfer chain of Rhodobacter sphaeroides
#Program in Biophysics and Computational Biology, University of Illinois at Urbana- Champaign, and *Department of Microbiology, University of Wisconsin, Madison
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.
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.
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