Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors FA and FB in Heliobacterium modesticaldum

The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates F-A- and F-B-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichia coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-F-X cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [F-A/F-B](-) were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798(+) and [F-A/F-B](-) reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the F-A- and F-B-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.