BChl Dimer Research Articles

Structure and function of cytochrome c2 in electron transfer complexes with the photosynthetic reaction center of Rhodobacter sphaeroides: optical linear dichroism and EPR.

The photosynthetic reaction center (RC) and its secondary electron donor the water-soluble cytochrome (cyt) c2 from the purple bacterium Rhodobacter sphaeroides have been used in cross-linked and non-cross-linked complexes, oriented in compressed gels or partially dried multilayers, to study the respective orientation of the primary donor P (BChl dimer) and of cyt c2. Three methods were used: (i) Polarized optical absorption spectra at 295 and 10 K were measured and the linear dichroism of the two individual transitions (Qx, Qy), which are nearly degenerate within the alpha-band of reduced cyt c2, was determined. Attribution of the polarization directions to the molecular axes within the heme plane yielded the average cyt orientation in the complexes. (ii) Time-resolved flash absorption measurements using polarized light allowed determination of the orientation of cyt c2 in complexes which differ in their kinetics of electron transfer. (iii) EPR spectroscopy of ferricyt c2 in cross-linked RC-cyt c2 complexes was used to determine the angle between the heme and the membrane plane. The results suggest the following structural properties for the docking of cyt c2 to the RC: (i) In cross-linked complexes, the two cytochromes displaying half-lives of 0.7 and 60 micros for electron transfer to P+ are similarly oriented (difference < 10 degrees). (ii) For cross-linked cyt c2 the heme plane is parallel to the symmetry axis of the RC (0 degrees +/- 10 degrees). Moreover, the Qy transition, which is assumed to be polarized within the ring III-ring I direction of the heme plane, makes an angle of 56 degrees +/- 1 degree with the symmetry axis. (iii) The dichroism spectrum for the fast phase (0.7 micros) for the non-cross-linked cyt c2-RC complex suggests an orientation similar to that of cross-linked cyt c2, but the heme plane is tilted about 20 degrees closer to the membrane. An alternative model is that two or more bound states of cyt c2 with heme plane tilt angles between 0 degrees and 30 degrees allow the fast electron transfer. Zero-length cross-linking of cyt c2 may take place in one of these bound states. These orientations of cyt c2 are compared to different structural models of RC-cyt c2 complexes proposed previously. The relation of the two kinetic phases observed in cross-linked cyt c2 complexes to biphasic kinetics of the mobile reaction partners is discussed with respect to the dynamic electrostatic interactions during the formation of a docking complex and its dissociation. A mechanism is proposed in which a pre-orientation of cyt c2 relative to the membrane plane occurs by interaction of its strong electrostatic dipole with the negative surface charges of the RC. The optimal matching of the oppositely charged surfaces of the two proteins necessitates further rotation of the cyt around its dipole axis.

Site-Directed Mutations Near the L-Subunit D-Helix of the Purple Bacterial Reaction Center: A Partial Model for the Primary Donor of Photosystem II

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Hydrogen-Bond Interactions of the Primary Donor of the Photosynthetic Purple Sulfur Bacterium Chromatium tepidum

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.

Excitation delocalization over the whole core antenna of photosynthetic purple bacteria evidenced by non-linear pump-probe spectroscopy

Anomalously high values of photoinduced absorption changes were revealed in the antenna of photosynthetic purple bacteria. They were found to be 4–16 times greater at the bleaching peak of the antenna than at the bleaching peak of the BChl dimer of the reaction center. This is direct proof of excitation delocalization over many pigment molecules. Calculations according to the model of exciton delocalization over all core antenna BChls allow one to explain the observed phenomenon.

THE EFFECT OF PERIPHERAL SUBSTITUTION ON THE BATHOCHROMIC SHIFT OF THE Qy TRANSITION OF BACTERIOCHLOROPHYLL DIMERS; in vitro MODELS OF THE PROTEIN EFFECT ON THE SPECTRUM OF PIGMENT CENTERS IN THE LIGHT‐HARVESTING COMPLEXES*

Abstract— The effect of chemical modifications in the side groups of the isocyclic ring V on the formation, optical absorption and circular dichroism of bacteriochlorophyll (Bchl) dimers was examined in a mixture of formamide and water containing TritonX–100 and variable amounts of pyridine. Substitution of the carbomethoxy group in the C132 position with a hydrogen atom, had no effect on the dimerization constant but increased the shift of the Qy transition by 1000 cm‐1 with respect to the native Bchl. Substitution of the C13 hydrogen atom with OH decreased the shift of the Qy transition by 400 cm‐1. The similarity between the spectra of the modified Bchl dimers and Bchl dimers in vivo indicates that protein binding to the side groups at Bchl dimers may profoundly affect the energy of their Qy transition but have minor effects on the Qy transitions of the monomelic Bchl.

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131.

Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhomogeneous spectral broadening of the B820 subunit form of LH1

Site-selected fluorescence spectra of the B820 subunit of LH1 from Rhodospirillum rubrum G9 were measured at temperatures between 160 K and 4.2 K. A linear correlation between excitation wavelength and maximal emission wavelength was observed across the whole Q y absorption band of the B820 subunits, which persists at higher temperatures. This demonstrates that (a) the Q y absorbance band of the B820 subunit is inhomogeneously broadened; (b) the subunit consists of a strongly interacting dimer of BChl a; (c) no energy transfer occurs among these dimers. The temperature dependence of the shape of the emission spectrum confirms that the protein forms a ‘glass-like’ environment for the bacteriochlorophyll pigments. After reassociating the octyl-glucoside-solubilized B820 subunits into the reassociated B873 form, the emission wavelength is independent of the wavelength of excitation at 4.2 K. This implies that the reassociated B873 form consists of a large number of interacting pigment molecules. The nature and origin of the spectral inhomogeneity in the B820 subunits is discussed.

Investigation into the source of electron transfer asymmetry in bacterial reaction centers

We have investigated the primary photochemistry of two symmetry-related mutants of Rhodobacter sphaeroides in which the histidine residues associated with the central Mg2+ ions of the two bacteriochlorophylls of the dimeric primary electron donor (His-L173 and His-M202) have been changed to leucine, affording bacteriochlorophyll (BChl)/bacteriopheophytin (BPh) heterodimers. Reaction centers (RCs) from the two mutants, (L)H173L and (M)H202L, have remarkably similar spectral and kinetic properties, although they are quite different from those of wild-type RCs. In both mutants, as in wild-type RCs, electron transfer to BPhL and not to BPhM is observed. These results suggest that asymmetry in the charge distribution of the excited BChl dimer (P*) in wild-type RCs (due to differing contributions of the two opposing intradimer charge-transfer states) contributes only modestly to the directionality of electron transfer. The results also suggest that differential orbital overlap of the two BChls of P with the chromophores on the L and M polypeptides does not contribute substantially to preferential electron transfer to BPhL.

Polypeptides and bacteriochlorophyll organization in the light-harvesting complex B850 of Rhodobacter sphaeroides R-26.1

The light-harvesting complex (LHC) B850 from Rhodobacter sphaeroides was dissociated into several fragments by treatment with sodium dodecyl sulfate. The molecular weight of each fragment was determined by using transverse polyacrylamide gel electrophoresis under nondenaturing conditions and gel filtration techniques. Four B850 LHCs were observed, having molecular weights of 60,000, 72,000-75,000, 105,000, and 125,000-145,000, and two small bacteriochlorophyll (Bchl)-polypeptide complexes having molecular weights of 6000-8000 and 12,000-14,000. Each of the B850 complexes contains ca. one Bchl a for each 6.5-kDa protein. The optical absorption and circular dichroism of the B850 LHCs recorded directly from the gels are similar to those measured previously for a 22-24-kDa B850 LHCs by Sauer and Austin [(1978) Biochemistry 17, 2011-2019]. These data, combined with studies of other groups, indicate that the smallest LHC in LH1 and LH2 is a Bchl-polypeptide tetramer. Each tetramer contains two Bchl dimers that probably have the structure of P-860, the primary electron donor in Rhodobacter sphaeroides, and two alpha-beta-polypeptide pairs. Interactions among the paired Bchls shift their individual Qy transitions from 780-800 to 850-860 nm, and interactions among two such pairs induce the circular dichroism signal of the LHCs. Three Bchl-polypeptide tetramers probably form a dodecamer having C3 symmetry, and six such dodecamers organize into a large hexagon that can accommodate one or two reaction center complexes.

Bacteriochlorophyll g epimer as a possible reaction center component of heliobacteria

Normal-phase HPLC analysis of acetone extracts of cells, membranes and antenna-reaction center complexes of Heliobacterium chlorum and Heliobacillus mobilis showed the presence of bacteriochlorophyll (BChl) g′, the 132-epimer of BChl g. The molar ratio of BChl g:BChl g′ in these preparations was approx. 18 in cells and membranes of both species. This value, when combined with a molar ratio of BChl g to the primary electron donor, P-798, of 35–40, yields a BChl g′:P-798 ratio of 2, suggesting that P-798 may be a dimer of BChl g′. The amount of BChl g′ in the antenna-reaction center complex of H. chlorum was slightly higher, suggesting that some epimerization may have occurred during the isolation of the complex. In contrast, bacteriopheophytins (BPhe) g and g′ were present in too small amouts to be essential components of the photosynthetic apparatus. This confirms the idea that the reaction center of heliobacteria is basically different from those of purple bacteria and Photosystem II. A detailed interpretation, based on Correlated Spectroscopy and Double Resonance experiments is given of the 1H-NMR spectra of BChl g and BChl g′, confirming the structure and identity of both pigments. Absorption and circular dichroism spectra of BChl g and BChl g′ are also presented.

Charge Separation in a Reaction Center Incorporating Bacteriochlorophyll for Photoactive Bacteriopheophytin

Site-directed mutagenic replacement of M subunit Leu214 by His in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides results in incorporation of a bacteriochlorophyll molecule (BChl) in place of the native bacteriopheophytin (BPh) electron acceptor. Evidence supporting this conclusion includes the ground-state absorption spectrum of the (M)L214H mutant, pigment and metal analyses, and time-resolved optical experiments. The genetically modified RC supports transmembrane charge separation from the photoexcited BChl dimer to the primary quinone through the new BChl molecule, but with a reduced quantum yield of 60 percent (compared to 100 percent in wild-type RCs). These results have important implications for the mechanism of charge separation in the RC, and rationalize the choice of (bacterio)pheophytins as electron acceptors in a variety of photosynthetic systems.

Effect of nonexcitonic interactions among the paired molecules on the Qy transition of bacteriochlorophyll dimers. Applications to the primary electron donors P-860 and P-960 in bacterial reaction centers

X-ray diffraction of reaction centers (RCs) from two strains of purple bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis, revealed that their primary electron donors, P-860 and P-960, are tight dimers of bacteriochlorophyll (Bchl) a and b, respectively. The geometry of the paired molecules is such that their transition monopoles strongly interact, resulting in a bathochromic shift of the Q{sub y} bands. Recent calculations based upon interactions among point-charge monopoles (PCM) showed that exciton coupling among the localized Q{sub y} transitions of the paired molecules can account for part of the shift. The origin of the remaining nonexcitonic shift is controversial. In this communication the authors show that there is a ratio of 1.2-1.5 between the nonexcitonic shift and the exciton splitting of the Q{sub y} transition in several Bchl dimers in vitro.

Efficiency of excitation trapping by reaction centres of complex B890 from Chromatium minutissimum

The formation and decay of excited states of light-harvesting bacteriochlorophyll (BChl) molecules and primary charge separation in reaction centres (RCs) of pigment-protein complex B890 of the purple bacterium Chromatium minutissimum were studied as a function of excitation wavelength by means of picosecond absorbance difference spectroscopy. Selective excitation in the RC absorption band (at 800 nm) induced rapid bleaching of the absorption band of the RC BChl dimer (τ ∼ 1 ps) without absorption changes due to excited light-harvesting BChl. It is concluded that excitation energy transfer from the BChl dimer of the RC to light-harvesting BChl molecules is considerably slower than RC charge separation. Therefore, energy transfer from the light-harvesting antenna to the RC in bacterial photosynthesis may be described as a ‘migration-limited model’.

Picosecond-photodichroism studies of the transient states in Rhodopseudomonas sphaeroides reaction centers at 5 K. Effects of electron transfer on the six bacteriochlorin pigments

We have examined the dichroism of the visible and near-infrared absorption changes due to the early transient states in Rhodopseudomonas sphaeroides reaction centers imbedded in polyvinyl alcohol films at 5 K. The transient-state, ground-state and derivative spectra acquired under these conditions are highly resolved. Spectral features have been assigned to the bacteriochlorophyll (BChl) dimer (P) that serves as the primary electron donor, to each of the two additional BChls, and to the two bacteriopheophytin (BPh) molecules. The dichroism of the absorption changes, taken together with earlier results including our observation of a detection-wavelength dependence of the kinetics, argues that only one of the BPhs is a clearly resolved electron carrier prior to ubiquinone. The second BPh and the two BChls not constituting P display electrochromic effects and/or nuclear relaxations, possibly involving the protein, in response to the charge-separation process.

Kinetics of electron transfer in duroquinone-reconstituted reaction centers from photosynthetic bacteria

Reaction centers isolated from photosynthetic bacteria are of interest both as a model system for biochemical electron transfer and as a simple system for study of the primary photochemistry of photosynthesis. The light-induced electron-transfer processes in purple photosynthetic bacteria begin with excitation of a bacteriochlorophyll (Bchl) dimer with maximum absorbance at 870 nm (P870). A relatively stable charge-separated state, consisting of oxidized P870 and reduced ubiquinone (UQ), is reached in < 1 ns [ 1,2]. An earlier unstable state almost certainly consists of P870+ and reduced bacteriopheophytin (BPh) [3]. Ubiquinone extraction leads to an apparent loss of photochemical activity than can be recovered by addition of UQ or a variety of other quinones [4-71. Here, we report experiments on reaction centers reconstituted with duroquinone (DQ, 2,3,5,6tetramethylbenzoquinone). We have found that the quantum yield of formation of the state P870+DQis essentially 1.0. The temperature dependence of the recombination kinetics of this state are anomalous, with temperature-independent regions at both low and high temperatures.

Subpicosecond and picosecond studies of electron transfer intermediates in Rhodopseudomonas sphaeroides reaction centers

The primary electron transfer processes in isolated reaction centers of Rhodopseudomonas sphaeroides have been investigated with subpicosecond and picosecond spectroscopic techniques. Spectra and kinetics of the absorbance changes following excitation with 0.7-ps 610-nm pulses, absorbed predominantly by bacteriochlorophyll (BChl), indicate that the radical pair state P +BPh −, in which an electron has been transferred from the BChl dimer (P) to a bacteriopheophytin (BPh), is formed with a time constant no greater than 4 ps. The initial absorbance changes also reveal an earlier state, which could be an excited singlet state, or a P +BChl − radical pair. The bleaching at 870 nm produced by 7 ps excitation pulses at 530 nm (absorbed by BPh) or at 600 nm (absorbed predominantly by BChl) shows no resolvable delay with respect to standard compounds in solution, suggesting that the time for energy transfer from BPh to P is less than 7 ps. However, the bleaching in the BPh band at 545 nm following 7-ps 600-nm excitation, exhibits an 8- to 10-ps lag with respect to standard compounds. This finding is qualitatively similar to the 35-ps delay previously observed at 760 nm by Shuvalov at al. (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Matveetz, Y.A. and Kryukov, P.G. (1978) FEBS Lett. 91, 135–139) when 25-ps 880-nm excitation flashes were used. A delay in the bleaching approximately equal to the width of the excitation flash can be explained in terms of the opposing effects of bleaching due to the reduction of BPh, and absorbance increases due to short-lived excited states (probably of BChl) that turn over rapidly during the flash. The decay of the initial bleaching at 800 nm produced by 7-ps 530- or 600-nm excitation flashes shows a fast component with a 30-ps time constant, in addition to a slower component having the 200-ps kinetics expected for the decay of P +BPh −. The dependence on excitation intensity of the absorbance changes due to the 30-ps component indicate that the quantum yield of the state responsible for this step is lower than that observed for the primary electron transfer reactions. This suggests that at least part of the transient bleaching at 800 nm is due to a secondary process, possibly caused by excitation with an excessive number of photons. If the 800-nm absorbing BChl (B) acts as an intermediate electron carrier in the primary photochemical reaction, electron transfer between B and the BPh must have a time constant no greater than 4 ps.