Cytochrome bc1-cy fusion complexes reveal the distance constraints for functional electron transfer between photosynthesis components

Abstract

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt c(y)) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-c(y) fusion complex that supported Ps growth solely relying on membrane-confined ET ( Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta 1757, 346-352 ). In this work, we further characterized this cyt bc1-c(y) fusion complex, and used its derivatives with shorter cyt c(y) linkers as "molecular rulers" to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-c(y) fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt c(y) linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt c(y) linker decreased drastically this electronic coupling between the cyt bc1-c(y) fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt c(y) linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-c(y) fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are "hardwired" for cyclic ET.

FIGURE 1.

A, amino acid sequence alignments of R. capsulatus cyt c_y and its shorter linker variants with its homologues in other species, R. capsulatus cyt c_y (native linker) (CAA79860); R. capsulaturs cyt c_y (Δ19); R. capsulatus cyt c_y (Δ24); R. sphaeroides cyt c_y (AAC26877); P. denitrificans cyt c_M (CAA49830); and S. pomeroyi DSS-3 cyt c₅₅₂ (AAV96763). B, hypothetical three-dimensional structural models of the R. capsulatus cyt bc₁-c_y fusion complex and its shorter linker derivatives. The cyt c domain of R. capsulatus c_y was modeled using SWISS-MODEL, and the overall structures were visualized using the R. capsulatus cyt bc₁ (Protein Data Bank 1ZRT) and yeast cyt bc₁:cyt c co-crystal (PDB 1NTK) structures.

FIGURE 2.

A, photosynthetic growth properties on liquid and solid media (enriched MPYE) of various R. capsulatus strains (YO2 lacking the cyt bc₁, cyt c_y, and cyt c₂; pMTS1/MT-RBC1 overproducing the cyt bc₁ complex, MT-G4/S4 lacking the cyt c₂, and pYO38/YO2, pYO33/YO2, and pYO30/YO2 containing the cyt bc₁-c_y fusion complexes with the native 19 amino acids and 24-amino acid shorter cyt c_y linkers, respectively) were determined by monitoring the turbidity of the cultures, as described under “Experimental Procedures.” B, the c-type cytochrome profiles of the same strains were revealed using chromatophore membranes (100 μg of total proteins per lane) and 15% SDS-PAGE/TMBZ analyses. C, DBH₂:cyt c reductase activities of chromatophore membranes (20 μg of total proteins) derived from the same strains described above were determined as in Atta-Asafo-Adjei and Daldal (20), in the absence (no inhibitor) or presence (10 μm stigmatellin or 20 μm antimycin), and for comparative purposes the steady-state enzymatic activities are represented as % of the overproduced native cyt bc₁ complex.

FIGURE 3.

Reduced minus oxidized optical difference spectra of b-type and c-type cytochromes in chromatophore membranes (0.4 mg of total proteins) from various R. capsulatus strains. Strains were as described in the legend to Fig. 2, and grown on enriched MPYE medium by respiration.

FIGURE 4.

EPR spectra of the cyt bc₁ complex [2Fe-2S] clusters (upper panel) and the cyt b hemes (lower panel) of R. capsulatus MT-G4/S4 lacking cyt c₂ and pYO38/YO2 containing a cyt bc₁-c_y fusion complex with a native cyt c_y linker, as described in the legend to Fig. 2. For EPR spectroscopy of the [2Fe-2S] cluster or cyt b hemes (b_L and b_H), chromatophore membranes were reduced with ascorbate or air oxidized, respectively, as described in Refs. and . Spectra were recorded at 20 K, 9.443 GHz, 12 G and 10 K, 9.59 GHz, 10 G temperature, microwave frequencies, and modulation amplitudes, respectively.

FIGURE 5.

Purification of the cyt bc₁-c_y fusion complex. A, SDS-PAGE, TMBZ, and immunoblot analyses. Approximately 50 μg of total proteins per lane were used in each case, except the pool from anti-FLAG, which had only 10 μg. Column fractions obtained during the purification procedure, and α-cyt c₁, α-FLAG (i.e. cyt c_y), α-cyt b, and α-Fe-S antibodies were as described under “Experimental Procedures” and in the text. B, size exclusion chromatography (upper panel) of the purified cyt bc₁-c_y fusion complex and cyt bc₁ complex in the presence of 150 mm NaCl and 0.05% DDM. Gel filtration chromatography was performed using a Superose 6 HR 10/30 column, which was run at a flow rate of 0.3 ml/min, and the elution profile was monitored at 280 nm. The column was calibrated with blue dextran (2,000 kDa), thyroglobulin (669 kDa), apoferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) as standards, run in the presence of 150 mm NaCl and 0.05% DDM, and their elution positions are indicated at the top of the chromatograph. Aliquots of each fraction (0.5 ml) were concentrated and subjected to 15% SDS-PAGE and immunoblot analyses (lower panel) using subunit-specific antibodies as described in A. C, dark equilibrium redox titration of the cyt c₁-c_y subunit of the purified cyt bc₁-c_y fusion complex (0.1 mg/ml). The titration was performed in 50 mm MOPS buffer (pH 7.0) containing 0.1 m KCl and 1 mm EDTA in the presence of 0.01% (w/v) DDM. Redox mediators were as described under “Experimental Procedures” (26). The E_m,7 value indicated was determined by fitting the normalized data to a n = 1 Nernst equation.

FIGURE 6.

Light-induced, time-resolved cyt b reduction and cyt c re-reduction kinetics of various R. capsulatus strains. In each case, chromatophore membranes containing an amount of RC equal to 0.45 μm were resuspended in 50 mm MOPS buffer (pH 7.0) containing 100 mm KCl and 100 mm EDTA at an E_h of 100 mV. The amount of RC was determined based on the extent of its photooxidation by a train of 10 flashes separated by 50 ms at an E_h of 380 mV, and using an extinction coefficient ε_605-540 of 29 mm^-1 cm^-1, as described under “Experimental Procedures.” The traces for cyt b reduction (upper panel) were monitored in the absence (No) and the presence of the Q_i site inhibitor antimycin (Ant, 5 μm), and those for cyt c re-reduction (lower panel) were in the absence of inhibitor or in the presence of myxothiazol (Myx, 5 μm), where no QH₂ oxidation takes place at the Q_o site, or in the presence of stigmatellin (Stig, 1 μm), where no electron is transferred from the [2Fe2S] cluster to the c₁ heme. All samples contained the same amounts of the cyt bc₁-c_y complexes as documented in Figs. 2 and 3.

FIGURE 7.

A, crystal structure of the RC-LH1 core complex (PDB 1PYH) from Rhodopseudomonas palustris and hypothetical three-dimensional structural model of R. capsulatus cyt bc₁-c_y fusion complex (pYO30/YO2, Δ24-c_y) (with the transmembrane helices of cyt bc₁ shown as ribbons, and cyt c_y (Δ24-c_y) shown as sticks), are drawn using the program PyMOL. The narrow section of the RC (subunits L, yellow; M, blue; H, purple) surrounded by the LH1 complex (chains α, pale green and β, green) and the cyt bc₁-c_y fusion complex (subunits cyt b, cyan; cyt c₁, orange; cyt c_y, red; and the Fe-S protein, magenta) are viewed parallel to the membrane plane. B, top view (perpendicular to the membrane plane) of the RC-LH1 core complex and the cyt bc₁-c_y fusion complex with the shortest cyt c_y linker (Δ24-c_y) is shown. Note that to reach the central part of RC subunits L and M in the RC-LH1 and cyt bc₁-c_y fusion complexes, the cyt c domain of cyt c_y (assuming that its linker is stretched out in a fashion parallel to the membrane) needs to reach out for about 100 Å. C, a schematic representation of the major membrane proteins involved in cyclic ET of purple bacterial photosynthesis (upper panel), and a proposed mechanism for cyt c_y-mediated cyclic ET via supraorganization of R. capsulatus photosynthetic unit (lower panel). cyt bc₁ complex, hydroquinone cyt c oxidoreductase; cyt c_y, membrane-associated cytochrome c_y. Arrows indicate directions of electron (e^-) and excitation (ex) flows.