Qy Transition Research Articles

Resonance Raman spectroscopy of metal‐substituted bacteriochlorophylls: characterization of Raman bands sensitive to bacteriochlorin conformation

Fourier transform (FT) preresonance Raman spectra of Mg-containing bacteriochlorophyll a (BChla) and the metal-substituted Zn-, Ni-, Cu- and Co-bacteriopheophytins a (BPhea) allowed the characterization of those vibrational modes sensitive to the nature of the central metal, through its effect on the core size of these molecules. Six of the Raman modes active in the excitation conditions used (1064 nm, in preresonance with the Qy transition of the metal–bacteriopheophytin molecules) are shown to be sensitive to the conformation of the dihydrophorbin ring. Comparison of the FT-Raman spectra of BChla under conditions where the central Mg ion is either five- or six-coordinate show that these modes may be used to distinguish modifications of the dihydrophorbin core size as small as 1 pm, and thus that they may be used for characterizing conformational strains on BChla molecules both in vitro and in vivo. © 1997 John Wiley & Sons, Ltd.

Energy Transfer in Rhodobacter sphaeroides Reaction Centers with the Initial Electron Donor Oxidized or Missing

Energy transfer between chromophores in the reaction center of Rhodobacter sphaeroides R-26 with and without the initial donor oxidized and in reaction centers from the strain VR(L157) that has the mutation Val to Arg at L157 has been investigated using femtosecond transient absorption spectroscopy. Pigment extractions and an analysis of the ground-state absorbance spectrum indicate that VR(L157) reaction centers have a substantial reduction in the amount of bacteriochlorophyll present per reaction center, due to the loss of one or both of the bacteriochlorophylls in the initial electron donor (P). The QY transition bands of the bacteriochlorophyll monomers (B) and the bacteriopheophytins (H) in reaction centers from the three reaction center samples (R-26 with and without P oxidized and VR(L157)) were selectively excited using 150 fs duration, 5 nm bandwidth pulses, and transient absorbance spectra were recorded. Quenching of the lowest excited state of the bacteriochlorophyll monomer (B*) occurs in hundreds of femtoseconds in R-26 reaction centers either with or without P initially oxidized. Selective excitation of B in VR(L157) reaction centers results in a nanosecond lifetime B* excited state. Neither charge separation nor fast quenching of B* is observed in VR(L157) reaction centers, contrary to what one might expect from predictions of the energetics of B* relative to charge-separated states such as B+H-. The fluorescence spectrum of B* in this mutant is similar in width to the absorbance spectrum of B and is centered near 801 nm (the mutant's ground-state B band peaks at 796 nm). Picosecond fluorescence measurements of VR(L157) reaction centers show that fluorescence decay from B* is multiexponential and occurs on the order of nanoseconds. From these results, it is concluded that P is required for fast quenching of B but that the oxidation state of P does not strongly affect the quenching process. This suggests that spectral or orbital overlap between B and P does not limit the ultrafast rate of energy transfer between these two cofactors. One possibility is that nuclear motion limits the rate of energy transfer.

Spectroscopy and structure of bacteriochlorophyll dimers. I. Structural consequences of nonconservative circular dichroism spectra

The origin of the nonconservative nature of the circular dichroism (CD) spectrum of bacteriochlorophyll dimers is investigated. It is shown that coupling between the Qy and Qx transitions can, under rather restricting circumstances, lead to an asymmetrical CD spectrum: only for a limited set of relative orientations of the monomers within the dimer is the spectrum found to be asymmetrical. The relation between intensity and asymmetry of the CD spectrum is elucidated. The results are applied to the B820 subunit of the LH1 antenna system and subsequently to the antenna system LH1 itself. Differences in the geometry of the BChls in LH1 versus the LH2 structure are discussed.

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.

The role of chromophore coupling in tuning the spectral properties of peripheral light-harvesting protein of purple bacteria

The publication of a structure for the peripheral light-harvesting complex of a purple photosynthetic bacterium (McDermott et al. (1995), Nature 374: 517-521) provides a framework within which we can begin to understand various functional aspects of these complexes, in particular the relationship between the structure and the red-shift of the bacteriochlorophyll Qy transition. In this article we describe calculations of some of the spectral properties expected for an array of chromophores with the observed geometry. We report the stability of the calculated absorption spectrum to minor structural alterations, and deduce that the observed red shift of the 850 nm Qy transition in the B800-850 antenna complexes is about equally attributable to chromophore-chromophore and chromophore-protein interactions, while chromophore-chromophore interactions predominate in generating the red-shift of the 820 nm Qy transition in B800-820 type peripheral liggt-harvesting complexes. Finally we suggest that the red shift in the absorbance of the monomeric Bchl a found in antenna complexes to 800 nm, from 770 nm as observed in most solvents, is largely attributable to a hydrogen bond with the 2-acetyl group of this chromophore.

Structure‐Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll‐Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light‐harvesting complex 2 (LH2) bacteriochlorophyll‐protein antenna complex from the purple non‐sulfur photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure‐based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near‐infrared region use a point‐monopole approximation to represent the BChl Qy transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane‐associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm−1, respectively) for the Qy transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm−1 wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.

The PSII-S protein of higher plants: a new type of pigment-binding protein.

An intrinsic 22 kDa protein of photosystem II has been shown to possess high sequence homology with the CAB gene products, but differs from these proteins by an additional putative fourth transmembrane helix. This protein, designated PSII-S in accordance with the assignment of the name psbS to its gene, has been isolated by nonionic detergents and preparative isoelectric focusing in this study. The isolated PSII-S protein was shown to bind 5 chlorophyll molecules (a and b) per protein unit and also several different kinds of carotenoids. The room temperature absorption spectrum of the Qy transition of the chlorophylls bound to the isolated protein is characterized by a broad band with a maximum at 671 nm. The 77 K fluorescence spectrum exhibits a peak at 672 nm. A single photon counting technique was applied to resolve the room temperature decay kinetics of the first excited singlet states in the chlorophyll ensemble of the PSII-S protein. The data can be satisfactorily described by triexponential kinetics with lifetimes of tau 1 = 1.8 ns, tau 2 = 4.4 ns, and tau 3 = 6.1 ns and normalized amplitudes of 0.09, 0.60, and 0.31, respectively. Circular dichroism spectra suggest that, in contrast to LHCII, virtually no pigment coupling exists in the PSII-S protein. Two copies of the PSII-S protein were found per PSII in spinach thylakoids. It displays an unusually extreme lateral heterogeneity, since the PSII beta centers located in the stroma exposed thylakoid regions contained only residual amounts of the PSII-S protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Femtosecond transient absorption spectroscopy on the light-harvesting Chl a/b protein complex of Photosystem II at room temperature and 12 K

The kinetics of excitation energy transfer processes within the light-harvesting Chl a/b protein complex of Photosystem II (LHC II) from spinach was investigated using femtosecond transient absorption difference spectroscopy at room temperature and 12 K. Upon excitation of Chl b with 640 nm pulses of 120 fs duration, time-resolved absorption changes were measured at different wavelengths within the Qy(0-0) transition band of Chl a. At room temperature the transient spectra showed a single negative band which is due to photobleaching and/or stimulated emission (PB/SE). The peak position of this band was found to be time independent (>-400 fs) at about 680 nm. In contrast, at 12 K the maximum of the PB/SE band shifted from 676 nm at 400 fs to about 680 nm at later times (⩾ 100 ps). The absorption difference spectra at 12 K showed a bipolarity at times ⩾ 7.7 ps, where excited state absorption and PB/SE predominate at shorter and longer wavelengths, respectively. The dynamic spectral evolution processes at 12 K could be rationalized in terms of excitation energy transfer between excitonically coupled antenna pigments in isolated LHC II complexes. Based on a global analysis of the flash induced absorption changes at 12 K several components have been obtained with lifetimes in the subpicosecond and picosecond time domain. The results of these fits are discussed with respect to the observed time dependent spectral changes.

Femtosecond pump-probe spectroscopy of bacteriochlorophyll a monomers in solution

One- and two-color absorption difference profiles were obtained for BChl a in 1-propanol with approximately 50-fs resolution, using a self-mode-locked Ti:sapphire laser system. Time evolution in the BChl a absorption difference spectrum produces nonexponential photobleaching/stimulated emission (PB/SE) decay kinetics in 800-nm one-color experiments. Nonexponential PB/SE rise behavior occurs for some combinations of pump and probe wavelengths in two-color experiments. Optimized parameters from triexponential fits to the absorption difference profiles depend markedly on the fitting time window; they typically include a minor component with lifetime in the hundreds of fs. Much of the latter component is due to vibrational relaxation and/or intramolecular vibrational redistribution, rather than solvent dielectric relaxation. Measurements of the pump-probe anisotropy indicate that the electronic transition moment for the broad Qy excited state absorption band that overlaps the Qy steady-state absorption spectrum makes an angle of at most 20 degrees from that of the ground-->Qy state transition. No coherent oscillations are observed at early times. Our results bear directly on the interpretation of fs pump-probe experiments on BChl a-containing pigment-protein complexes.

Femtosecond photodichroism studies of isolated photosystem II reaction centers.

Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 beta-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Qy transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.

Protochlorophyllide forms in non‐greening epicotyls of dark‐grown pea (Pisum sativum)

Low‐temperature fluorescence emission spectra of 6.5‐day‐old dark‐grown epicotyls of pea (Pisum sativum) revealed the presence of protochlorophyll(ide). The upper part of the epicotyl contained 30% of the protochlorophyll(ide) content per fresh weight found in pea leaves, whereas the lower part contained 3%. Three discrete spectral forms of protochlorophyll(ide) were clearly distinguished after Gaussian deconvolution of fluorescence excitation and emission spectra. Adding the satellite bands of the Qy(0‐0) transitions (the emission vibrational (Emv) bands with correlated amplitudes, gave the following delineation: Ex439–Em629–Emv684, Ex447–Em636–Emv700 and Ex456–Em650–Emv728. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by immunodetection of whole tissue extracts of the epicotyl indicated the presence of NADPH‐protochlorophyllide oxidoreductase (EC 1.3.1.33). Electron micrographs showed prolamellar bodies in at most 11 % of the plastid profiles of the epicotyl cells. These prolamellar bodies were smaller, and many of them showed less regular structure than those of the leaves. Taken together, the results indicate that the protochlorophyll(ide) in epicotyls is arranged in a different way than in leaves.

Core Antenna Complexes, CP43 and CP47, of Higher Plant Photosystem II. Spectral Properties, Pigment Stoichiometry, and Amino Acid Composition

The core antenna complexes of photosystem II, CP43 and CP47, were purified from two higher plants by anion-exchange chromatography, using a combination of the chaotropic agent LiClO4 and the nonionic detergent beta-dodecyl maltoside. The Qy transition was resolved at 48 K into two main bands near 682.3 and 671.5 nm for CP43, while the CP47 spectrum showed a more complex structure with main bands at 688, 681.2, 676, 667, and 661 nm. Emission bands (77 K) were detected at 683 and 695 nm for CP43 and CP47, respectively. Fluorescence excitation spectra showed high efficiency of energy transfer between the different transitions of the chlorophylls and a somewhat lower efficiency from beta-carotene. The circular dichroism spectrum of CP47 indicated the presence of excitonic interactions between some chlorophylls. In contrast, CP43 showed a single negative circular dichroism band at 670 nm. The pigment content of the complexes was determined by both spectroscopic measurements and HPLC. Contents of 18 chlorophylls a and 5 beta-carotenes per CP43 polypeptide and 19 chlorophylls a and 3 beta-carotenes per CP47 polypeptide were found, using the methods of Lowry or Bradford for protein quantitation. When the protein concentration was determined from the amino acid analysis, 20 chlorophylls a and 5 beta-carotenes per CP43 and 21-22 chlorophylls a and 4 beta-carotenes per CP47 were obtained. Thus, a content of 46-48 chlorophylls a was obtained for the core complex, assuming 4-6 chlorophylls per reaction center, in agreement with the composition obtained experimentally using a highly purified oxygen-evolving core complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized fluorescence and absorption of macroscopically aligned Light Harvesting Complex II

Polarized absorption and fluorescence measurements have been performed at 77 K on isotropic and anisotropic preparations of trimeric Light Harvesting Complex II (LHC-II) from spinach. The results enable a decomposition of the absorption spectrum into components parallel and perpendicular to the trimeric plane. For the first time, it is shown quantitatively that the strong absorption band around 676 nm is polarized essentially parallel to the plane of the trimer, i.e., the average angle between the corresponding transition dipole moments and this plane is at most 12 degrees. The different absorption bands for LHC-II should not be considered as corresponding to individual pigments but to collective excitations of different pigments. Nevertheless, the average angle between the Qy transition dipole moments of all chlorophyll a pigments in LHC-II and the trimeric plane could be determined and was found to be 17.5 degrees +/- 2.5 degrees. For the chlorophyll b pigments, this angle is significantly larger (close to 35 degrees). At 77 K, most of the fluorescence stems from a weak band above 676 nm and the corresponding transition dipole moments are oriented further out of plane than the dipole moments corresponding to the 676-nm band. The results are shown to be of crucial significance for understanding the relation between the LHC-II structure and its spectroscopy.

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.

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Qy transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Qy transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.

Analysis of the 77 K fluorescence emission and excitation spectra of isolated etioplast inner membranes

Low-temperature fluorescence emission and excitation analyses revealed four discrete spectral forms of protochlorophyllide and/or protochlorophyll in the etioplast inner membranes isolated from leaves of wheat ( Triticum aestivum cv. Kosack). The emission spectra were recorded with various excitation wavelengths between 400 and 500 nm. The excitation spectra were recorded for fluorescence emitted at various wavelengths between 620 and 740 nm. The spectra were analysed by a combination of various computer-aided methods including differential analysis, gaussian resolution and topological plots. The four pigment forms can be characterized by their different excitation (Ex) and emission (Em) bands. Adding the satellite bands of the Qy (0-0) transitions, the emission vibrational (Emv) bands, with correlated amplitudes gave the following delineation: Ex436-Em633-Emv690, Ex443-Em645-Emv711, Ex451-Em657-Emv727, and Ex463-Em671-Emv740. The differences in the spectral properties of the four pigment forms are discussed in relation to possible differences in their structural arrangement, association to NADPH-protochlorophyllide oxidoreductase and localization in prolamellar bodies and/or prothylakoids.

Steady-state polarized light spectroscopy of isolated Photosystem I complexes

Monomeric and trimeric Photosystem I core complexes from the cyanobacterium Synechocystis PCC 6803 and LHC-I containing Photosystem I (PS I-200) complexes from spinach have been characterized by steady-state, polarized light spectroscopy at 77 K. The absorption spectra of the monomeric and trimeric core complexes from Synechocystis were remarkably similar, except for the amplitude of a spectral component at long wavelength, which was about twice as large in the trimeric complexes. This spectral component did not contribute significantly to the CD-spectrum. The (77 K) steady-state emission spectra showed prominent peaks at 724 nm (for the Synechocystis core complexes) and at 735 nm (for PS I-200). A comparison of the excitation spectra of the main emission band and the absorption spectra suggested that a significant part of the excitations do not pass the red pigments before being trapped by P-700. Polarized fluorescence excitation spectra of the monomeric and trimeric core complexes revealed a remarkably high anisotropy (∼0.3) above 705 nm. This suggested one or more of the following possibilities: 1) there is one red-most pigment to which all excitations are directed, 2) there are more red-most pigments but with (almost) parallel orientations, 3) there are more red-most pigments, but they are not connected by energy transfer. The high anisotropy above 705 nm of the trimeric complexes indicated that the long-wavelength pigments on different monomers are not connected by energy transfer. In contrary to the Synechocystis core complexes, the anisotropy spectrum of the LHC I containing complexes from spinach was not constant in the region of the long-wavelength pigments, and decreased significantly below 720 nm, the wavelength where the long-wavelength pigments on the core complexes start to absorb. These results suggested that in spinach the long-wavelength pigments on core and LHC-I are connected by energy transfer and have a non-parallel average Qy(0-0) transitions.

A structural role of the carotenoid in the light-harvesting II protein of Rhodobacter capsulatus

The membrane-linked light-harvesting II protein (LHII) of Rhodobacter capsulatus was partly depleted of carotenoids by selective extraction with light petroleum. Carotenoid removal was accompanied by bleaching of the Qy(S1<--S0) absorption band of bacteriochlorophyll (Bchl) a near 800 nm, by a bathochromic shift and a broadening of the other Bchl Qy band at 850 nm, and by the formation of a weak Qy band of dissociated Bchl near 770 nm. The changes in the 800 and 850 nm bands seemed to reflect alterations in only those Bchl molecules that had lost their associated carotenoids, firstly, because the extent of the changes was closely correlated to the degree of carotenoid extraction, and, secondly, because the residual fraction of carotenoid-containing LHII, which could be almost quantitatively recovered from the membrane after detergent solubilization and ion-exchange chromatography, showed an unmodified LHII absorption spectrum. The Bchl responsible for the shifted 850 nm band remained bound to protein, since its visible (Qx) transition seemed to retain the induced optical activity of the native bound pigment. Besides, the shifted Bchl could act as an efficient acceptor of singlet excitation energy from the pigments of the intact LHII fraction. The close similarity between the spectroscopic Bchl changes that accompany carotenoid extraction and the differential spectral features of carotenoidless LHII of Rhodobacter mutants, previously reported, strongly suggests that the direct cause of the spectral modifications is the absence of carotenoid and not any independent effect of the experimental manipulation of the membrane. Several interpretations of the structural changes that underlie the observed spectral changes are possible. The simplest one is to assume that carotenoid removal elicits an alteration in the angle between the Qy transition moments of two strongly interacting Bchl molecules.

THE MAJOR INTRINSIC LIGHT‐HARVESTING PROTEIN OF Amphidinium: CHARACTERIZATION AND RELATION TO OTHER LIGHT‐HARVESTING PROTEINS

A major light-harvesting complex (LHC) has been obtained from thylakoids of Amphidinium carterae solubilized with digitonin or decylmaltoside and separated by sucrose-gradient centrifugation. The digitonin-LHC forms a dark brown band at approximately 17% sucrose and the decylmaltoside LHC one at approximately 7% sucrose. Excellent energy transfer is retained from chlorophyll c and carotenoid to chlorophyll a. Absorbance and fluorescence excitation spectra show the existence of two major forms of chlorophyll c, one absorbing at 634 nm and the other at 649 nm. Linear dichroism spectra show the Qy transition of both forms of chlorophyll c to be aligned at < 35 degrees to the membrane plane. On sodium dodecylsulfate polyacrylamide gels the complex resolves as a single band of 19 kDa. A partial amino acid sequence shows the N-terminus to be unblocked but modified; there is a persistent ambiguity of Ser/Asn at residue 4 and evidence for multiple but very similar polypeptides within the 19 kDa band. The peptide has strong identity with the N-terminal regions of LHC from Phaeodactylum and Pavlova and LHC 1 of higher plants. Antibodies to the 19 kDa peptide react weakly with LHC of brown algae, diatoms and Prymnesiophytes but not with those of higher plants or Cryptophytes.

SIMULATIONS OF Prosthecochloris BACTERIOCHLOROPHYLL a‐PROTEIN OPTICAL SPECTRA IMPROVED BY PARAMETRIC COMPUTER SEARCH

AbstractRecently, reasonable spectral simulations of the experimental low‐temperature absorption and circular dichroism (CD) spectra of the bacteriochlorophyll (BChl) a protein from Prosthecochloris aestuarii were produced for the unaggregated protein trimer based on standard assumptions regarding QY transition moment directions. We report here significant improvements of these simulations, obtained by computer search of unknown parameters including the site wavelengths of the individual BChl and exciton‐transition line widths. Absorption and CD spectra are fit with a common set of search parameter values. As before, calculated exciton‐transition wavelengths and line widths also compare favorably with values deduced from laser hole‐burning experiments. However, the improved simulations are only obtained when comparison is made to 77 K data obtained with a cryosolvent containing glycerol but not glycerophosphate. Simulations of 77 K data obtained with a mixture of glycerol and glycerophosphate are poorer than those recently reported. (The latter are based on 5 K absorption and 77 K CD data, with transition line widths independently selected for each simulation.) Computer tests of the search routine demonstrate that these results are independent of the routine itself. A possible explanation is that the glycerophosphate‐containing cryosolvent perturbs the protein structure enough to alter slightly the inter‐BChl geometry.