Complex Of Rhodobacter Sphaeroides Research Articles

Three-Dimensional Structure of the Rhodobacter sphaeroides RC-LH1-PufX Complex: Dimerization and Quinone Channels Promoted by PufX

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

Proton environment of reduced Rieske iron-sulfur cluster probed by two-dimensional ESEEM spectroscopy.

The proton environment of the reduced [2Fe-2S] cluster in the water-soluble head domain of the Rieske iron-sulfur protein (ISF) from the cytochrome bc(1) complex of Rhodobacter sphaeroides has been studied by orientation-selected X-band 2D ESEEM. The 2D spectra show multiple cross-peaks from protons, with considerable overlap. Samples in which (1)H(2)O water was replaced by (2)H(2)O were used to determine which of the observed peaks belong to exchangeable protons, likely involved in hydrogen bonds in the neighborhood of the cluster. By correlating the cross-peaks from 2D spectra recorded at different parts of the EPR spectrum, lines from nine distinct proton signals were identified. Assignment of the proton signals was based on a point-dipole model for interaction with electrons of Fe(III) and Fe(II) ions, using the high-resolution structure of ISF from Rb. sphaeroides. Analysis of experimental and calculated tensors has led us to conclude that even 2D spectra do not completely resolve all contributions from nearby protons. Particularly, the seven resolved signals from nonexchangeable protons could be produced by at least 13 protons. The contributions from exchangeable protons were resolved by difference spectra ((1)H(2)O minus (2)H(2)O), and assigned to two groups of protons with distinct anisotropic hyperfine values. The largest measured coupling exceeded any calculated value. This discrepancy could result from limitations of the point dipole approximation in dealing with the distribution of spin density over the sulfur atoms of the cluster and the cysteine ligands, or from differences between the structure in solution and the crystallographic structure. The approach demonstrated here provides a paradigm for a wide range of studies in which hydrogen-bonding interactions with metallic centers has a crucial role in understanding the function.

An alternative carotenoid-to-bacteriochlorophyll energy transfer pathway in photosynthetic light harvesting.

Blue and green sunlight become available for photosynthetic energy conversion through the light-harvesting (LH) function of carotenoids, which involves transfer of carotenoid singlet excited states to nearby (bacterio)chlorophylls (BChls). The excited-state manifold of carotenoids usually is described in terms of two singlet states, S(1) and S(2), of which only the latter can be populated from the ground state by the absorption of one photon. Both states are capable of energy transfer to (B)Chl. We recently showed that in the LH1 complex of the purple bacterium Rhodospirillum rubrum, which is rather inefficient in carotenoid-to-BChl energy transfer, a third additional carotenoid excited singlet state is formed. This state, which we termed S*, was found to be a precursor on an ultrafast fission reaction pathway to carotenoid triplet state formation. Here we present evidence that S* is formed with significant yield in the LH2 complex of Rhodobacter sphaeroides, which has a highly efficient carotenoid LH function. We demonstrate that S* is actively involved in the energy transfer process to BChl and thus have uncovered an alternative pathway of carotenoid-to-BChl energy transfer. In competition with energy transfer to BChl, fission occurs from S*, leading to ultrafast formation of carotenoid triplets. Analysis in terms of a kinetic model indicates that energy transfer through S* accounts for 10-15% of the total energy transfer to BChl, and that inclusion of this pathway is necessary to obtain a highly efficient LH function of carotenoids.

The Interaction of the Rieske Iron-Sulfur Protein with Occupants of the Qo-site of the bc1 Complex, Probed by Electron Spin Echo Envelope Modulation

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.

Stark Hole-Burning Studies of Three Photosynthetic Complexes

Stark hole-burning spectroscopy at 1.8 K was used to determine the dipole moment changes fΔμ (f, the local field correction factor) for the B800 absorption band of the light harvesting 2 (LH2) complex of Rhodobacter sphaeroides, Rhodopseudomonas acidophila (strain 10050), and Rhodospirillum molischianum. Hole-burning values of fΔμ for the lowest energy exciton level (B870) associated with LH2's B850 band have recently been reported (Rätsep et al. Spectrochim. Acta, in press). Values for the lowest energy exciton level (B896) associated with the B875 band of the LH1 complex of Rb. sphaeroides (wild-type chromatophores and an LH1-only mutant) and the 825 nm band of the bacteriochlorophyll a (FMO) antenna complex of Chlorobium tepidum are also reported. For each band, fΔμ was determined for burn laser polarization parallel and perpendicular to the Stark field ES and several burn frequencies. The dependencies on laser polarization and burn frequency are typically quite weak. Importantly, fΔμ values for the above bands are small, falling in the range ∼0.5−1.2 D, with the lowest and highest values associated with the 825 nm band of the FMO complex and B800 band of the LH2 complex, respectively. For the B896 band of the LH1 complex, fΔμ ≈ 0.8 D. Such small values are consistent with the very weak linear electron−phonon coupling of antenna protein complexes as determined by hole-burning spectroscopy. Overall, the values for fΔμ from classical Stark modulation (CSM) studies (Gottfried et al. Biochim. Biophys. Acta 1991, 1059, 63; Beekman et al. J. Phys. Chem. B 1997, 101, 7293) are larger, in the cases of B850 and B875, by a factor of 3−4. (In CSM spectroscopy, one analyzes the response of the entire absorption band to the external field.) Discussion of the discrepancies between the two Stark techniques is given. It appears that difficulties inherent to the analysis procedure of CSM spectroscopy can lead to unreliable values for dipole moment and polarizability changes associated with absorption bands of photosynthetic complexes, especially when several excitonic levels contribute to the band, e.g., B850 and B875. An explanation for the small hole-burning values of fΔμ for the B870 and B896 levels associated with Cn cyclic arrays of strongly coupled BChl a dimers is given based on structural, symmetry, and energy disorder considerations. A key point is that, in the absence of energy disorder, the component of Δμj (j labeling the exciton level) perpendicular to the Cn axis is zero. Energy disorder, which destroys the Cn symmetry and leads to localization effects results in nonzero values which may depend on j when the protein-induced contribution to Δμj is taken into account.

Low-intensity pump-probe spectroscopy on the B800 to B850 transfer in the light harvesting 2 complex of Rhodobacter sphaeroides

The isolated LH2 (B800–850) complex of Rb. sphaeroides has been studied at 77 K using low-intensity one-colour pump-probe spectroscopy. Delta absorbance transients were measured at several wavelengths within the B800 band. In the red part of the band, the excited B800 population decays mono-exponentially with a lifetime of about 1.2 ± 0.1 ps. More to the blue much faster rates are found which are ascribed to downhill energy-transfer among the B800 pigments. To our surprise, increasing the average intensity from 4 W/cm 2 to higher excitation densities significantly slows the decay of B800 ∗. Since this effect is permanent we conclude that it is due to some form of photodamage. We propose that this observation explains the slower decay reported by others.

Fluorescence spectroscopy of 13′‐cis‐spheroidene at 170 K: A reason for the natural selection of the all‐trans configuration for the light‐harvesting function

AbstractAs a follow‐up to fluorescence and fluorescence‐excitation spectroscopy at 170 K of all‐trans‐spheroidene free in solutionsY. Watanabe, T. Kameyama, Y. Miki, M. Kuki, and Y. Koyama, “The 2′Ag− state and two additional low‐lying electronic states of spheroidene newly identified by fluorescence and fluorescence‐excitation spectroscopy at 170K,” Chem. Phys. Lett. 206, 62–68 (1993). and bound to the light‐harvesting complex of Rhodobacter sphaeroides 2.4.1,Y. Koyama, Y. Miki, T. Kameyama, R. J. Cogdell, and Y. Watanabe, “Low‐lying electronic levels of spheroidene bound to the light‐harvesting (LH2) complex of Rhodobacter sphaeroides 2.4.1. as determined by fluorescence and fluorescence‐excitation spectroscopy at 170K,” Chem. Phys. Lett. 208, 479–485 (1993). 13′‐cis‐spheroidene in n‐hexane solution has been examined for comparison. All‐trans‐spheroidene exhibits efficient internal conversion from the 3A to the 2A state and fluorescence from both the 2A and B states, while 13‐‐cis‐spheroidene exhibits internal conversion from the “3Ag‐” state to the “B” state and fluorescence only from the “B” state. Thus, all‐trans‐spheroidene can provide two channels of carotenoid‐to‐bacteriochlorophyll singlet‐energy transfer, while 13′‐cis‐spheroidene can provide only one. This must be a reason for the natural selection of the all‐trans configuration for the light‐harvesting function. © 1995 John Wiley & Sons, Inc.

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 μs for the B800-850 complex, 14±2 μs for the B850 complex incorporated with spheroidene, and 19±2 μs for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 μs. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49-55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.

Low-lying electronic levels of spheroidene bound to the light-harvesting (LH2) complex of Rhodobacter sphaeroides 2.4.1 as determined by fluorescence and fluorescence—excitation spectroscopy at 170 K

Fluorescence and fluorescence—excitation spectroscopy at 170 K of the light-harvesting (LH2) complex of Rhodobacter sphaeroides 2.4.1 has determined the energies of level I (2 1A g −, level III ( 1B u +) and level IV of the bound spheroidene, as the absorptive 0 ← 0 transitions, to be 14500, 18300, 19200 and 23200 cm −1, respectively. Internal conversion in spheriodene and spheroidene-to-bacteriochlorophyll energy transfer, as determined by fluorescence—excitation spectroscopy, suggest two different light-harvesting pathways: (1) capturing the light energy and transferring the singlet energy through level III, and (2) capturing the light energy via level IV and transferring the rest of the singlet energy via level I, after internal conversion (energy dissipation) from level IV to level I.

Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.

EPR characterization of the cytochrome b- c1 complex from Rhodobacter sphaeroides

EPR characteristics of cytochrome c 1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b- c 1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c 1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b- c 1 complex are: g x = 1.8 with a small shoulder at g = 1.76, g y = 1.89 and g z = 2.02, similar to those observed with the mitochondrial enzyme. The g x = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the g z and and shifts it downfield from g = 2.02 to 2.03, and shifts g x upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of g y but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the g y from 1.89 to 1.94 and broadens the g x signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the g y and g z signals, but they broaden and shift the g x signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b- c 1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV. The concentration of antimycin sensitive Q-radical correlates well with the enzymatic activity of the complex. The antimycin-sensitive ubisemiquinone radical exhibits a pH dependency of approx. 60 mV / pH unit, between pH 8.0 and 9.0, suggesting the possible involvement of a dissociable group, with a pK a between 8.0 and 9.0, in Q-binding.