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The cyanobacterium, Synechocystis PCC 6803, with a histidine
(His)-tag attached to the carboxy terminus of the-PSII chlorophyll
a-binding protein, CP47 (ref. 7), was grown photoheterotrophically
in the presence and absence of iron in the culture medium.-PSI fractions
free of-PSII (seeMethods) were resolved by sucrose density centrifugation.
In the case of normal cells, the sucrose gradient separated two
chlorophyll a-containing fractions, corresponding to-PSI monomers
and-PSI trimers. In addition to these fractions, the iron-stressed
cells produced two extra bands, one less dense than the-PSI monomer
and one more dense than the-PSI trimer. -polyacrylamide gel
electrophoresis (SDS-PAGE; see Methods) showed (Fig. 1) the less
dense of the iron-stress-induced fractions to be free CP43' (confirmed
by immunoblotting). This technique also showed the more dense fraction
to be a CP43'-PSI complex consisting of the PsaA/PsaB reaction
centre subunits of-PSI (PSI RC, with a relative molecular mass of
75,000:Mr 75K), the CP43' protein and other lower-Mr-PSI proteins.
HPLC size exclusion chromatography gave an estimated Mr of approximately
1,900K for the CP43'-PSI complex, compared with 1,000K for the-PSI
trimer.
In Fig. 2 we show room-temperature optical absorption spectra, and
77K emission spectra, of-PSI trimers, free CP43' and the CP43'-PSI complexes. Although the absorption spectrum of the CP43'-PSI complex
had a blue-shifted, long-wavelength absorption maximum compared
with that of the-PSI trimer due to the presence of CP43', the 77K
fluorescence spectrum of the CP43'-PSI complex gave the same maximum
as that of the-PSI trimer alone. As isolated CP43' was highly fluorescent
with a maximum at 685 nm, we conclude that in the CP43'-PSI complex,
CP43' is associated with-PSI in such away as to allow efficient energy
transfer. The small level of fluorescence at 685 nm from the CP43'-PSI complex seen in Fig. 2b can be attributed to some unattached CP43'
in the preparation. Excitation spectra for the 720-nm emission from
the CP43'-PSI complex confirmed that most of the CP43' present efficiently
transferred energy to-PSI.
To gain a better understanding of the association of CP43' with-PSI,
we conducted single-particle analysis of the CP43'-PSI complex on
images obtained by electron microscopy. Figure 3a and b show typical
electron micrographs of-PSI trimers and the novel CP43'-PSI complex
stained in uranyl acetate. In both cases, mainly top views were
seen, although some stacked side views were present. Top-view averages
of the-PSI trimers and the CP43'-PSI complex are shown in Fig. 3c
and d at a resolution of about 25 Angstrom . The-PSI trimer (Fig.
3c) corresponds in size and organization to that seen previously
by electron microscopy8,9 and is consistent with X-ray diffraction
studies on the-PSI trimer of the thermophilic cyanobacterium Synechococcus
elongatus 10. The CP43'-PSI complex shown in Fig. 3d is more
circular, with a diameter of about 330 Angstrom corresponding to
a 55 Angstrom ring of CP43' subunits around the-PSI trimer. The
thicknesses of the-PSI trimer and CP43'-PSI trimer supercomplex
are approximately the same, as seen in Fig. 3a and b, and estimated
to be about 90 Angstrom . This indicates that the additional ring
of CP43' in the CP43'-PSI complex is contained within the membrane.
Image processing suggested that the ring was composed of 18 copies
of CP43', and that the top views were probably of the stromal surface9,10.
The 4 Angstrom X-ray structure of the cyanobacterial-PSI trimer has been
determined10. Moreover, there is a 3.8 Angstrom structural model of-PSII
isolated from S. elongatus11, which, as suggested from electron
crystallography12, shows CP43 to be a ring of three pairs of transmembrane
helices. In Fig. 4 we have modelled the X-ray structures of the-PSI trimer and CP43 into the projection map of the CP43'-PSI trimer
supercomplex. This modelling suggests that there are indeed 18 copies
of CP43' per supercomplex. Given that CP43 binds at least 12 chlorophyll
molecules within its six-helix bundle11, we conclude that the additional
antenna size of the ironstress- induced CP43'-PSI supercomplex is
composed of approximately 216 chlorophyll a molecules. As the-PSI
trimer contains about 300 chlorophyll molecules13, then the light-harvesting
ability of the supercomplex has increased by approximately 72% compared
with that of the normal trimer. This increase in antenna size is
almost certainly in response to the reduction in the level of the
lightharvesting phycobilisomes and-PSI complexes due to the heavy
demand on iron for their synthesis and assembly3. Among the various
hypotheses for the physiological role of CP43', it has been suggested
that it may act as an additional light-harvesting system for-PSII
given that CP43' is a-PSII-like protein3. Its functional association
with-PSI is therefore surprising. The formation of a ring of light-harvesting
chlorophyll binding proteins around a reaction centre is reminiscent
of the organization of the antenna systems of anoxygenic purple
photosynthetic bacteria14,15.We have shown that similar rings can
occur in oxygenic photosynthetic organisms.
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