Identification and Characterization of PshB, the Dicluster Ferredoxin that Harbors the Terminal Electron Acceptors F_A and F_B in Heliobacterium modesticaldum^,

Abstract

The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates FA- and FB-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-FX 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 [FA/FB]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798+ and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-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.

The Genome of Heliobacterium modesticaldum , a Phototrophic Representative of the Firmicutes Containing the Simplest Photosynthetic Apparatus

“…In addition to pshA, a pshB gene (HM1_1462), which encodes an RC-associated [4Fe-4S]-binding ferredoxin, was confirmed to be present in the H. modesticaldum genome. A recent study showed that PshB binds the terminal Fe/S dicluster electron acceptors, F A and F B (19). Electrons are likely transferred from the Fe/S clusters to a cytoplasmic ferredoxin, which can supply reducing equivalents for cellular processes, such as carbon assimilation and nitrogen fixation (Fig.…”

The bound iron–sulfur clusters of Type-I homodimeric reaction centers

“…This effort was aided by an earlier report of the isolation and amino acid sequences of two bacterial dicluster ferredoxins, Fd1 and Fd2, from H. mobilis (Hatano et al 2005). The N-terminal amino acid sequence of Fd2 was seen to be similar to the N-terminal sequence of PshBI from H. modesticaldum (Heinnickel et al 2007). After cloning the pshBI gene (annotated as HM1_1462 in H. modesticaldum) and identifying its location on the H. modesticaldum genome, it became clear that fd2 and pshBI are orthologs (Heinnickel et al 2007).…”

“…The N-terminal amino acid sequence of Fd2 was seen to be similar to the N-terminal sequence of PshBI from H. modesticaldum (Heinnickel et al 2007). After cloning the pshBI gene (annotated as HM1_1462 in H. modesticaldum) and identifying its location on the H. modesticaldum genome, it became clear that fd2 and pshBI are orthologs (Heinnickel et al 2007). It is interesting to recall that a similar situation occurred during the early days of PsaC, in which the psaC gene in chloroplasts was initially annotated as fdx1, a bacterial dicluster ferredoxin.…”

“…PsaC also contains an eight amino acid insertion between the Fe/S cluster binding motifs that interacts with ferredoxin and flavodoxin (Naver et al 1998). A sequence comparison of PshBI and PsaC shows that both of these features are missing in PshBI (Heinnickel et al 2007).…”

Heliobacterial Rieske/cytb complex

“…Different preparation procedures suggested either an F A /F B -type protein (Lin et al 1995;Nuijs et al 1985;Heinnickel et al 2006Heinnickel et al , 2007 or a quinone as electron acceptor (Trost and Blankenship 1989). The quinone present in the membrane is menaquinone with MK9 as the dominant form (Trost and Blankenship 1989;Hiraishi 1989).…”