Unifying principles in homodimeric type I photosynthetic reaction centers: Properties of PscB and the F_A, F_B and F_X 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 F_A^- and F_B^- and were unable to reduce NADP⁺. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the F_A and F_B 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 Fe^3+,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 F_X^-. 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 F_A and F_B iron-sulfur clusters are present on a salt-dissociable subunit, and F_X is present as an interpolypeptide [4Fe-4S]^2+,1+ cluster with a significant population in a S = 3/2 ground spin state.