Structural and spectroscopic properties of a reaction center complex from the chlorosome-lacking filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii

Abstract

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.

FIG. 1.

Pigmented bands in preparative PAGE after anion-exchange chromatography (A) and near-infrared absorption spectra of two pigment bands containing bacteriochlorophylls (B). Two pigmented bands (I and II) were extracted in 10 mM Tris buffer (pH 8.7) containing 0.05% Triton X-100.

FIG. 2.

Absorption spectra (A) and oxidized-minus-reduced difference spectra (B) of the RC preparation of R. castenholzii. The reduced form (solid line in panel A) was prepared by the addition of a small amount of Na₂S₂O₄, and the oxidized form (dashed line in panel A) was prepared by addition of K₃Fe(CN)₆.

FIG. 3.

Flash-induced difference absorbance and spectral change of cytochrome c in the prepared photochemical reaction center of R. castenholzii. (A) A kinetic trace showing the flash-induced absorbance change at 553 to 540 nm. The trace is an average of 60 measurements at intervals of 45 s. (B) A corresponding transient spectrum at 20 ms after the actinic flash. The content of RC was adjusted to 0.003 μM. The buffer contained 20 mM MOPS-KOH (pH 7.0), 2 mM sodium ascorbate, and the redox mediators vitamin K₃ (10 mM) and vitamin K₁ (30 mM).

FIG. 4.

Polypeptide composition of the purified RC-LH complex. Seven micrograms of protein of the RC-LH complex was denatured by boiling for 7 min in the presence of 1% β-mercaptoethanol. After electrophoresis (15% acrylamide gel), the gel was stained with Coomassie brilliant blue (CBB) and a heme-staining reagent (Heme).

FIG. 5.

Nucleotide and deduced amino acid sequences of the region of the pufLM gene encompassing the C-terminal region of the L subunit and the amino-terminal region of the M subunit of R. castenholzii. The putative ribosome-binding sites are shaded. Boldface indicates the amino acids identified by automated Edman sequencing of the purified polypeptides. The putative additional transmembrane helix is underlined. Numbers indicate the distance from the first nucleotide of the pufB gene.

FIG. 6.

Alignment of the amino acid sequences of the M subunit in the RC complex. The upper two organisms belong to the filamentous anoxygenic phototrophic bacteria. The lower three organisms are purple bacteria. Consensus amino acid residues for pigment ligands are boxed. Identical amino acids are indicated by asterisks.

FIG. 7.

Comparison of the primary structures of the cytochrome subunits of R. castenholzii (R. cas), C. aurantiacus (C. aur), and B. viridis (B. vir). Consensus amino acid residues of heme-binding motifs are boxed, and axial ligands to the heme irons are shaded and indicated by the heme numbers. Identical amino acids are indicated by asterisks.

FIG. 8.

Phylogenetic trees showing the relationships of RC subunits of purple bacteria and filamentous anoxygenic phototrophic bacteria. Trees were constructed from the deduced amino acid sequences of the L and M subunits (A) and cytochrome subunit (B). Bootstrap values from 1,000 replicates are indicated at the branching points. The accession numbers of the sequences used to construct the trees are as follows: C. aurantiacus, X14979 and AF288462; R. sphaeroides, AJ010302; Rhodobacter capsulatus, Z11165; Rubrivivax gelatinosus, AB034704; B. viridis, X03915 and X05768; Roseateles depolymerans, AB028938; Roseobacter denitrificans, X83392; Rhodovulum sulfidophilum, AB020784; Acidiphilium rubrum, AB005218.