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

Abstract

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 alpha/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 microseconds 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-microseconds 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 FX and FAFB, 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 t1/2 = 20 ms that we suggest occurs between Fx- and P840+. Degradation of the iron-sulfur clusters resulted in replacement of the 20-ms phase with a faster reaction of t1/2 = 80 microseconds that was most likely a recombination between the early acceptor A1- and P840+ or decay of 3P840. 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Redox Titration of Two [4Fe‐4S] Clusters in the Photosynthetic Reaction Center from the Anaerobic Green Sulfur Bacterium Chlorobium vibrioforme

“…Similar spectra have been observed in C. limicola f. sp. thiosulfatophilum (Nitschke et al, 1990;Feiler et al, 1992;Oh-oka et al, 1993) and C. vibrioforme (Miller et al, 1992) and C. tepedum (Kusumoto et al, 1994). In analogy with PS I, these resonances (termed signal 111) are proposed to represent an interaction spectrum from two spin coupled iron-sulfur clusters within the same protein.…”

“…In two landmark studies, Knaff and Malkin (1976) identified a low potential iron-sulfur center with a midpoint potential of approximately -550 mV in chromatophores from C. limicola f. thiosulfatophilum, and Jennings and Evans (1977) described the irreversible, light-induced reduction of a component with a midpoint potential of approximately -550 mV in a preparation from the same organism. Typically, a set of resonances with a trough at about g = 1.85 are seen on illumination at cryogenic temperatures in C. limicola f. thiosulfatophilum (Jennings and Evans, 1977;Nitschke et al, 1990;Feiler et al, 1992;Oh-oka et al, 1993), C. vibrioforme (Kjcer et al, 1994) and C. tepidum (Kusumoto et al, 1994), and a spectrum with mid-field and high-field g values of 1.89 and 1.91 is observed when samples are frozen in light to photoaccumulate more than one electron in C. limicola f. thiosulfatophilum (Nitschke et al, 1990;Feiler et al, 1992;Oh-oka et al, 1993), C. vibrioforme (Kjcer et al, 1994;Miller et al, 1992) and C. tepidum (Kusumoto et al, 1994). A similar spectrum is found in chemically reduced reaction centers of C. tepidum (Kusumoto et al, 1994).…”

Two molecules of cytochrome c function as the electron donors to P840 in the reaction center complex isolated from a green sulfur bacterium, Chlorobium tepidum

“…(i) A pair of PscA core protein, that shows homology in amino acid sequences to both of PsaA and PsaB core proteins of PS I, seem to make up the so-called 'homodimer' RC in contrast to the asymmetric 'heterodimer' RC of other photosynthetic organisms [1]. (ii) The RC contains three iron-sulfur clusters that resemble Fx, FA and F B clusters in PSI RC [2][3][4][5][6]. The binding motifs for F x-and FAIFB-like clusters reside in the PscA core and the PscB proteins, respectively, in C. limicola [1,7].…”

“…The evidence for the secondary acceptor quinone is still scarce. Photo-oxidized P840 is reduced by cytochrome c [3,5,10,11]. The gene encoding the cytochrome c identified in C. vihrioforme has a single heine binding moiety in a conserved motif, Cys-X-X-Cys-His [8].…”

The electronic structure of P840+• The primary donor of the Chlorobium limicola f. sp. thiosulphatophilum photosynthetic reaction centre

“…Such a spectrum can be obtained for PSI, but is only observed in PSI1 or purple bacteria if the non-heme iron is removed. The observation of an electron spin polarized signal [8,10], and indirect evidence suggesting that the triplet of P840' is affected by conditions that could lead to double reduction of a quinone [7] suggest the presence of an 'AI' quinone acceptor in C thiosulphatophilum. However, the presence of a quinone acceptor in Heliobacteriaceae is disputed [ 111.…”