Abstract
Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.
Highlights
Bearing membrane proteins [1]
We found that cyt soluble variant of cyt cy (S-cy) variants support native-like Ps growth provided that they are present at sufficient amounts in vivo
Molecular genetic analyses described under “Experimental Procedures” revealed that revertants R3 and R5 contained plasmid-borne Cys to Thr and Ala to Gly base pair substitutions in cycY, respectively, and produced cyt S-cyR3 (His at position 53 substituted with Tyr) and cyt S-cyR5 (Lys at position 19 substituted with Arg) variants of cyt S-cy
Summary
Chromatophore membranes used for SDS-PAGE/3,3Ј,5,5Ј-tetramethylbenzidine (TMBZ) or light-activated kinetic spectroscopy analyses were prepared in 50 mM MOPS buffer, pH 7.0, 1 mM phenylmethylsulfonyl fluoride dissolved in dimethyl sulfoxide and containing either 1 or 100 mM KCl, respectively [9]. Chemical reduction-oxidation midpoint potential (Em,7) determination of supernatant fractions of chromatophore membranes were performed according to Dutton [27] in 50 mM MOPS, 100 mM KCl, pH 7.0, in the presence of 15 to 30 M of redox mediators (tetrachlorohyroquinone, 2,3,5,6-tetramethyl-1,4-phenylenediamine, 1,2-naphthoquinone-4-sulfonate, 1,2-naphthoquinone, phenazine ethosulfate, phenazine methosulfate, duroquinone, pyocyanine, 2-hydroxy-1,4-naphthoquinone, and anthraquinone-2-sulfonate) as described earlier [8]. Light-activated, millisecond time scale kinetic spectroscopy was performed using chromatophore membranes reduced with sodium ascorbate, as described earlier [8, 9]. The carotenoid band shift was monitored at 490 – 475 nm to follow the generation of the transmembrane potential by cyclic ET [8, 9]
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