A New Cytochrome Subunit Bound to the Photosynthetic Reaction Center in the Purple Bacterium, Rhodovulum sulfidophilum

Abstract

The nucleotide sequence of the puf operon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ, pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense that pufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from the pufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1-binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the "tetraheme" cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.

A New Membrane-bound Cytochrome c Works as an Electron Donor to the Photosynthetic Reaction Center Complex in the Purple Bacterium, Rhodovulum sulfidophilum

“…1, where the heme-protein content of such intact cells was examined by SDS-PAGE and specific staining. Bands with apparent molecular masses around 30 and 46 kDa were assigned to the cytochrome c 1 of the cytochrome bc 1 complex and to the RC-bound cytochrome subunit, respectively, in agreement with a previous study (9). An intense band attributable to both cytochrome c 2 and cytochrome c-556 was detected at around 14 kDa.…”

“…Such molecular building blocks seem widely conserved, because these four heme-binding motifs have been reported even in green filamentous bacteria, Chloroflexus aurantiacus and Roseiflexus castenholzii, which are rather distant from purple bacteria (7,8). On the other hand, among purple bacteria themselves, in Rhodovulum sulfidophilum, the amino acid sequence of the RC-bound cytochrome subunit lacks one of the heme-binding motifs (9). Moreover, the methionine 6th-axial-ligand to one of the hemes is replaced by a cysteine residue (9,10).…”

“…On the other hand, among purple bacteria themselves, in Rhodovulum sulfidophilum, the amino acid sequence of the RC-bound cytochrome subunit lacks one of the heme-binding motifs (9). Moreover, the methionine 6th-axial-ligand to one of the hemes is replaced by a cysteine residue (9,10). The missing heme in Rvu.…”

Structural and Functional Characterization of the Unusual Triheme Cytochrome Bound to the Reaction Center of Rhodovulum sulfidophilum

“…In typical purple bacterial tetraheme cytochromes, heme 2(IV) has a His/His ligation pattern and consequently displays a low redox potential. The conservation of the axial ligands in the sequence of the R. sulfidophilum triheme cytochrome (9) suggests that this heme has a similarly low E m value also in the triheme cytochrome. The ϩ30 mV, g ϭ 3.12 heme is therefore attributed to heme 2(IV).…”

“…Construction of Plasmids and Site-specific Mutagenesis-The whole puf operon, containing genes coding for the L, M, and C (cytochrome) subunits of the RC complex and the ␣ and ␤ subunits of the lightharvesting complex of R. sulfidophilum was cloned (9). The NaeI-SphI fragment containing the 3Ј-region of pufM, the pufC gene, and the 5Ј-region of open reading frame 641 was cut out and inserted into plasmid pUC118 at the SmaI-SphI sites, yielding pRSS113.…”