Part 4 Results

Isolation of the CP43'-PSI Supercomplex-To isolate the CP43-PSI supercomplex from Synechocystis PCC6803, we used a mutant that had a His tag attached to the C terminus of CP47, kindly provided by Dr. T. Bricker (Louisana State University, Baton Rouge) (18). The mutant was grown photoheterotrophically in the presence and absence of iron in the culture medium. Thylakoid membranes were isolated, and after solubilization with 1% beta-D-dodecyl maltoside, were passed through a Ni2+ affinity column. PSII was selectively bound to the column via the His tag, whereas the non-bound fraction containing PSI was collected and subjected to sucrose density gradient centrifugation. Fig. 2a shows that in the case of normal cells (Gradient A) two main chlorophyll-containing bands were observed corresponding to monomeric (band 2) and trimeric (band 3) PSI, whereas iron-stressed cells (Gradient B) gave two additional green bands (bands 1 and 4). The SDS-polyacrylamide gel electrophoresis analysis shown in Fig. 2b characterized the various bands and revealed that bands 1 and 4 contained free CP43' and CP43' plus PSI respectively. Size exclusion high performance liquid chromatography analysis of the solubilized PSI fractions presented in Fig. 3a indicated that the two PSI bands obtained with normal cells corresponded to the approximate molecular masses expected for a monomeric (~356 kDa) and trimeric (1068 kDa) PSI complex (32) with the trimer being the dominant species. The additional peaks observed after iron stress correspond to native chlorophyll-binding CP43' (~47 kDa) and a high molecular mass chlorophyllcontaining species of approximately 1900 kDa, indicative of a CP43' and PSI supercomplex. Also of importance is that the level of the PSI trimer in iron-stressed cells was significantlyreduced compared with that of normal cells when normalized against the monomeric level of PSI.

Spectral Characterization-The room temperature optical absorption spectra of the isolated PSI trimer, CP43', and the CP43'-PSI supercomplex are shown in Fig. 4. The PSI trimer has a long wavelength absorption maximum at 680 nm as compared with 670 nm for isolated free CP43'. As expected, the CP43'-PSI band has a maximum absorption at the intermediate wavelength of 673 nm. The high level of absorption in the 450-500-nm region in the case of CP43' is because of its copurification with free carotenoid in the sucrose density gradients (see asterisk in Fig. 2a). Fluorescence measured at 77 K showed that the PSI trimer had an emission maximum at 720 nm, whereas CP43' fluoresced maximally at 685 nm (Fig. 5a). However, in the case of the CP43'-PSI supercomplex, the emission profile was similar to that of PSI with the exception of some weak emission at approximately 685 nm. Upon the addition of 0.1% Triton X-100, this weak signal at 685 nm changed to the dominant emission (Fig. 5b), indicating that the detergent had uncoupled CP43' from PSI and therefore suggesting that in the untreated sample, energy is efficiently transferred from CP43' to PSI. Sucrose density gradient analyses showed that indeed the Triton X-100 treatment converted the CP43'-PSI band into trimeric PSI and free CP43' (data not shown). Further confirmation that CP43' within the CP43'-PSI supercomplex was functionally coupled to PSI was made by measuring excitation spectra for 77-K fluorescence emission at 720 nm (data not shown).