Abstract
The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene. This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria. The RC complex of C. tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD. PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria. We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function. 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis. 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities. 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells. The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840). 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain. The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I. The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.
Highlights
The PscD subunit in the homodimeric “type I” photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene
These structures have clarified that the folding motifs of membrane-spanning ␣-helices within core proteins as well as the spatial configurations of the electron transfer components are essentially identical in type I and II RCs, suggesting that they have a common origin
The RC complex of the green sulfur bacterium Chlorobium tepidum consists of four subunits, PscA, PscB, PscC, and PscD with an antenna size of about 30 bacteriochlorophyll (BChl) molecules, whereas the heterodimeric photosystem I complex is made of 12 polypeptides with a much larger antenna size of nearly 100 chlorophyll (Chl) molecules [4, 5]
Summary
Growth Conditions—The strain of C. tepidum used in this study was WT2321 [22]. For liquid cultivation and plating incubation, CL and CP media were prepared, respectively, as previously described [21]. The anaerobic growth on CP plates was carried out in an anaerobic jar (model G, code HP31; OXOID, Ltd.) equipped with a palladium catalyst and a H2/CO2-generating gas pack (BBL Gas Pack; BD Biosciences) and supplemented with 0.1 g of thioacetoamide to generate H2S by adding 1 ml of 0.5 M HCl. Plasmid Construction and Transformation of C. tepidum—A 1.2-kb fragment containing the pscD gene of C. tepidum was amplified by PCR, using a pscdF primer (5Ј-CTGAATTCAGTGGTACGAGAAGGCCATCC) and a pscdR primer (5Ј-CGGAATTCCGCTTGGCTGCAATTGCATCG), and cloned into the EcoRI site of pUC18, yielding plasmid pCD1. The photomultiplier was protected by a 340-nm interference filter with a bandwidth of 10 nm
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