Unifying principles in homodimeric type I photosynthetic reaction centers: Properties of PscB and the FA, FB and FX iron–sulfur clusters in green sulfur bacteria

The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from FA− and FB− and were unable to reduce NADP+. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the FA and FB clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. Mössbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe–4S]2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe–2S]2+,1+ clusters and rubredoxin-like Fe3+,2+ centers, and that the [4Fe–4S]2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g = 5.4 and 4.4 assigned to a S = 3/2 ground spin state [4Fe–4S]1+ cluster and at g = 1.77 assigned to a S = 1/2 ground spin state [4Fe–4S]1+ cluster, both from FX−. These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the FA and FB iron–sulfur clusters are present on a salt-dissociable subunit, and FX is present as an interpolypeptide [4Fe–4S]2+,1+ cluster with a significant population in a S = 3/2 ground spin state.