Photosynthetic Electron-Transfer Reactions in the Green Sulfur Bacterium Chlorobium Vibrioforme: Evidence for the Functional Involvement of Iron-Sulfur Redox Centers on the Acceptor Side of the Reaction Center

The green sulfur bacterium Chlorobium vibrioforme was cultured in the presence ofethylene to selectively inhibit the synthesis ofthe chlorosome antenna BChl d. Use of these cells as starting material simplified the isolation of a photoactive antenna-depleted membrane fraction without the use of high concentrations of detergents. The preparation had a BChl a/P₈₄₀ of 50, and the spectral properties were similar to those ofpreparations isolated from cells grown with a normal complement ofchlorosomes. The membrane preparation was active in NADP+ photoreduction. This indicated that the fraction contained reaction centers with complete electron-transfer sequences which were then characterized further by flash kinetic spectrophotometry and EPR. We confirmed that cytochrome c₅₅₃ is the endogenous donor to P₈₄₀⁺, and at room temperature we observed a recombination reaction between the reduced terminal acceptor and P₈₄₀⁺ with a t_1/2 = 7 ms. Oxidative degradation ofiron-sulfur centers using low concentrations ofchaotropic salts introduced a faster recombination reaction of t_1/2 = 50 μs which was lost at higher concentrations of chaotrope, indicating the participation of another iron-sulfur redox center earlier than the terminal acceptor. Cluster insertion using ferric chloride and sodium sulfide in the presence of 2-mercaptoethanol restored both the 50-μs and 7-ms recombination reactions, allowing definitive assignments of these centers as iron-sulfur centers. Following the suggestion ofNitschke et al. [(1990) Biochemistry 29, 3834-3842], we associate these two kinetic phases to back-reactions between P840⁺ and iron-sulfur centers F_x and F_AF_B, respectively. The iron-sulfur cluster degradation and reconstitution protocols also led to inhibition and restoration of NADP+ photoreduction by the membrane preparation, providing unequivocal evidence for the function of the centers Fx and FAFB in the physiological electron-transfer sequence on the acceptor side of the Chlorobium reaction center. At 77 K we observed a recombination reaction of t_1/2 = 20 ms that we suggest occurs between F_x^- and P₈₄₀⁺. Degradation of the iron-sulfur clusters resulted in replacement of the 20-ms phase with a faster reaction of t_1/2 = 80 μs that was most likely a recombination between the early acceptor Af and P₈₄₀⁺ or decay of ³P₈₄₀. Analysis of the iron-sulfur centers in the preparation by EPR atcryogenic temperature supports the optical measurements. EPR signals originating from the terminal acceptor(s) were not observed following treatment of the membrane preparation by chaotropes, and a modified signal was restored following cluster reinsertion. The reinsertion ofiron-sulfur clusters in the Chlorobium preparation did not require the addition of any polypeptides, and the inclusion of psaC and psaD gene products that are required for the reconstitution of photosystem 1 did not increase the extent ofthe Chlorobium reconstitution. Kinetic interactions between the redox centers are discussed with reference to the model ofthe Chlorobium reaction center proposed by Nitschke et al. [(1990) Biochemistry 29, 3834-3842].