Stable photobleaching of P840 in Chlorobium reaction center preparations: Presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide

Abstract

Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated. The subunit composition was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551. The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated. In any case, the major portion of the reaction center was photoactive in the preparations. A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show the tendency to form a complementary subcomplex. FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.

Function of a PscD Subunit in a Homodimeric Reaction Center Complex of the Photosynthetic Green Sulfur Bacterium Chlorobium tepidum Studied by Insertional Gene Inactivation

“…Chlorosome-free membranes were prepared, and the molar ratio of BChl a to P840 was estimated on the basis of the oxidized minus reduced difference spectra, as summarized in Table II. The ratio of BChl a/P840 in the membranes of wild-type cells was estimated to be 49, in agreement with previous reports (12,36,37), whereas the ratio obtained in the ⌬pscD membranes was 43. These results suggested that the FMO proteins were partially detached from the mutant membranes during the preparation and that the low efficiency in the energy transfer between FMO protein and RC would reflect some distorted organization of FMO protein.…”

“…PscD of C. tepidum, 143 residues; PsaD of Synechocystis sp. PCC 6803, 141 residues) and share similar distributions of charged basic residues, as previously mentioned by Hager-Braun et al (12). This is in clear contrast to the case that the amino acid sequences of F A /F B and core proteins show more divergent aspects between Chlorobium RC and photosystem I.…”

“…This accomplishment will be expected to become an initial step for a study on the energy conversion mechanism in the homodimeric RC. Since the amino acid sequences of PscD have been shown to retain a weak similarity to those of the PsaD of photosystem I, the former function had been speculated to stabilize the Chlorobium RC (12). However, the absence of PscD did not raise any alteration in the contents of other constituent subunits of the RC complex (Fig.…”

“…Three-dimensional imaging analysis with the electron microscopic technique (STEM) suggested that PscD was in contact with the Fenna-MatthewsOlson (FMO) protein on the cytoplasmic side (11). Although PscD has been assumed to function in the same way as PsaD in photosystem I, as judged from their sequence similarities (12), there is no definite evidence for this speculation. The PsaD of photosystem I has been demonstrated to stabilize F A /F B -containing PsaC and facilitate the electron transfer reaction to ferredoxin (Fd).…”

Membrane proteome analysis of the green‐sulfur bacterium Chlorobium tepidum

“…Our attempts were to improve the resolution of 2-DE by separation of the associated chlorosomes on the membranes. In contrast to other nonionic detergents, Triton X-100 was highly effective for the solubilization of the membranes of C. tepidum and the separation of the chlorosomes as aggregates [23]. Optimal results were obtained using the protocol as described in [15].…”