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 of ethylene to selectively inhibit the synthesis of the 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 of preparations isolated from cells grown with a normal complement of chlorosomes. 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 t1/2 = 7 ms. Oxidative degradation of iron-sulfur centers using low concentrations of chaotropic salts introduced a faster recombination reaction of t1/2 = 50-mu-s 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-mu-s and 7-ms recombination reactions, allowing definitive assignments of these centers as iron-sulfur centers. Following the suggestion of Nitschke 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(A)F(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 F(X) and F(A)F(B) in the physiological electron-transfer sequence on the acceptor side of the Chlorobium reaction center. At 77 K we observed a recombination reaction of t1/2 = 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 t1/2 = 80-mu-s that was most likely a recombination between the early acceptor A1- and P840+ or decay of P-3(840). Analysis of the iron-sulfur centers in the preparation by EPR at cryogenic 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 of iron-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 of the Chlorobium reconstitution. Kinetic interactions between the redox centers are discussed with reference to the model of the Chlorobium reaction center proposed by Nitschke et al. [(1990) Biochemistry 29, 3834-3842].