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We isolated the photosynthetic reaction center
from membranes of Heliobacterium modesticaldum
(HbRC) using >-dodecyl-b-D-maltopyranoside
followed by sucrose density ultra-centrifugation. The low
temperature EPR spectra of whole cells, isolated membranes, and
HbRC complexes are similar, showing a single Fe/S cluster with g-values
of 2.067, 1.933 and 1.890 after illumination at 20 K, and a complex
spectrum attributed to exchange interaction from two Fe/S clusters
after illumination during freezing. We removed the protein containing
the Fe/S clusters by washing the HbRC with 1.0 M NaCl and purified
by ultrafiltration over a 30-kDa cut-off membrane. Analysis of the
filtrate by SDS PAGE showed a major band at ~8 kDa that was
weakly stain with Coomassie Brilliant Blue and stronglystained with silver. The optical spectrum of
the oxidized Fe/S protein shows a maximum at 410 nm and the EPR
spectrum of the reduced Fe/S protein shows a complex set of resonances
similar to those found in 2[4Fe-4S] ferredoxins. We were able to
purify the HbRC core by DEAE ion exchange chromatography and found
that it was composed of a band at ca. 40 kDa, which is identified
as PshA, and several additional proteins. We then found that the
isolated Fe/S protein rebinds spontaneously to purified HbRC cores,
and the light-induced EPR signals of the Fe/S clusters can be recovered.
The flash-induced kinetics of the HbRC complex show two kinetic
phases at room temperature, one with a lifetime of 75 ms and the
other with a lifetime of 15 ms. The 75 ms component is lost when
the Fe/S protein is removed from the HbRC complex, and it is regained
when the Fe/S protein is rebound to HbRC cores. Thus, the 75 ms
kinetic phase is derived from recombination of a terminal Fe/S cluster
with P798+, and the 15 ms kinetic phase is derived from
recombination with an earlier acceptor, probably FX.
We suggest that the bound Fe/S protein present in the HbRC be designated
PshB.
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Figure 2.3.2.EPR spectr of salt-washed
embranes from Heliobacterium modesticaldum. The membranes were solubilized using n-dodecyl-b-D-maltopyranoside
and were either untreated(a) or treated with (b) 100 mM, (c) 250 mM or (d)
500 mM NaCl for 5 min.
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