Anisotropy of the emission and absorption bands of spinach chloroplasts measured by fluorescence polarization and polarized excitation spectra at low temperature

Spectra of fluorescence polarization were measured between 4 and 120 K of spinach chloroplasts, oriented in a magnetic field. At least seven emission bands were observed. The well known bands near 685 nm (‘F-685’) and 735–740 nm (‘F-735’) and the band near 680 nm (‘F-680’) were strongly polarized parallel to the plane of the thylakoid membrane, whereas emission bands near 695 nm (‘F-695’), 710, 730–735 and 760 nm showed perpendicular polarization. Assuming perfect orientation of the thylakoid membranes, we calculated orientation angles of 64, 47 and 66.5° for the emission dipoles of F-685, F-695 and F-735, respectively, with respect to the normal of the membrane. Excitation spectra of F-695 and F-735 in polarized light at 4 K provided information about the orientation of the absorption dipoles of chlorophylls a and b. The spectra thus obtained were in very good agreement with the linear dichroism spectrum. Moreover, they allowed us to distinguish between the pigments associated with Photosystems I and Ii, which is not possible from measurement of linear dichroism alone. The results indicate that a high degree of orientation is not confined to the long-wave absorbing bands, but also bands at shorter wavelength show a clear anisotropy. The calculated orientations were in quantitative agreement with the hypothesis that F-685 and F-735 are associated with chlorophylls absorbing at 676 and 710–715 nm, respectively.     

Chlorophyll transition dipole moment orientations and pathways for flow of excitation energy among the chlorophylls of the major plant antenna, LHCII

We have attempted in this work an assignment of the Qy dipole moment orientations for all the chlorophylls in the major plant antenna, light-harvesting complex II (LHCII). Information that has recently become available through a structural model of the LHCII, site-directed mutagenesis, and spectroscopy of both LHCII and CP29 has been evaluated to model the electronic excited state structure in the presence of chlorophyll-chlorophyll and chlorophyll-protein interactions. An assignment has been obtained which satisfactorily reproduces the polarized linear absorption characteristics. The assignment proposed has also been found to be adequate in reproducing the time scales of the energy transfer processes. The pathways for the flow of excitation energy among the chlorophylls of the complex have been suggested in the context of identity and orientation assignments.     

Light harvesting in photosystem I: modeling based on the 2.5-A structure of photosystem I from Synechococcus elongatus

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the F?rster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.     

Calculation of spectra of absorption and circular dichroism of Rhodopseudomonas acidophila light harvesting complexes based on roentgen structural data

Absorption and circular dichroism spectra of two forms of the peripheral light-harvesting complex from photosynthetic purple bacteria Rhodopseudomonas acidophila were calculated. Calculations were carried out on the basis of exciton theory for circular aggregates of bacteriochlorophyll molecules and X-ray data for these forms of the complex. It was shown that theoretical spectra fit well experimental ones at the same values of excitation energy, homogeneous and inhomogeneous broadening, and bandwidth for all bacteriochlorophyll molecules of complexes. To approximate the circular dichroism spectra of complexes, it was necessary to change the orientations and the values of the moments of transition of Qy molecules relative to their orientation determined on the basis of X-ray structure analysis data.     

Minor complexes at work: light-harvesting by carotenoids in the photosystem II antenna complexes CP24 and CP26

Plant photosynthesis relies on the capacity of chlorophylls and carotenoids to absorb light. One of the roles of carotenoids is to harvest green-blue light and transfer the excitation energy to the chlorophylls. The corresponding dynamics were investigated here for the first time, to our knowledge, in the CP26 and CP24 minor antenna complexes. The results for the two complexes differ substantially. In CP26 fast transfer (80 fs) occurs from the carotenoid S₂ state to chlorophylls a absorbing at 675 and 678 nm, whereas transfer from the hot S₁ state to the lowest energy chlorophylls is observed in <1 ps. In CP24, energy transfer from the S₂ state leads in 80 fs to the population of chlorophylls b and high-energy chlorophylls a absorbing at 670 nm, whereas the low-energy chlorophylls a are populated only in several picoseconds. The results suggest that CP26 has a structural and functional organization similar to that of LHCII, whereas CP24 differs substantially from the other Lhc complexes, especially regarding the lutein L1 binding domain. No energy transfer from the carotenoid S₁ state to chlorophylls was observed in either complex, suggesting that this state is energetically below the chlorophyll Qy state and therefore may play a role in the quenching of chlorophyll excitations.     

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.     

Electrooptical analysis of alpha-chymotrypsin at physiological salt concentration

The electric dichroism of alpha-chymotrypsin has been measured in a buffer containing 0.1 M Na(+), 10 mM Mg(2+) and 25 mM Tris-cacodylate pH 7.2. The reduced dichroism as a function of the electric field strength can be represented by the orientation function for permanent dipoles and is not consistent with the orientation function for induced dipoles. After correction for the internal directing field, the dipole moment is 1.1 x 10(-27) Cm (+/- 10%), corresponding to 340 D, at 20 degrees C. The assignment of the permanent dipole moment is confirmed by the shape of the dichroism rise curves, which require two exponentials with amplitudes of opposite sign for fitting. The dichroism decay time constants measured in the range of temperatures between 2 and 30 degrees C indicate a temperature induced change of the structure, which is equivalent to an increase of the hydrodynamic radius from r = 26.6 A at 2 degrees C to 28.5 A at 30 degrees C. Our results demonstrate that electrooptical investigations of proteins with a high time resolution can be extended to physiological salt concentrations without serious problems by use of appropriate instruments.     

Energy transfer among CP29 chlorophylls: calculated Förster rates and experimental transient absorption at room temperature

The energy transfer rates between chlorophylls in the light harvesting complex CP29 of higher plants at room temperature were calculated ab initio according to the F?rster mechanism (F?rster T. 1948, Ann. Physik. 2:55-67). Recently, the transition moment orientation of CP29 chlorophylls was determined by differential linear dichroism and absorption spectroscopy of wild-type versus mutant proteins in which single chromophores were missing (Simonetto R., Crimi M., Sandonà D., Croce R., Cinque G., Breton J., and Bassi R. 1999. Biochemistry. 38:12974-12983). In this way the Q(y) transition energy and chlorophyll a/b affinity of each binding site was obtained and their characteristics supported by reconstruction of steady-state linear dichroism and absorption spectra at room temperature. In this study, the spectral form of individual chlorophyll a and b ligands within the protein environment was experimentally determined, and their extinction coefficients were also used to evaluate the absolute overlap integral between donors and acceptors employing the Stepanov relation for both the emission spectrum and the Stokes shift. This information was used to calculate the time-dependent excitation redistribution among CP29 chlorophylls on solving numerically the Pauli master equation of the complex: transient absorption measurements in the (sub)picosecond time scale were simulated and compared to pump-and-probe experimental data in the Q(y) region on the native CP29 at room temperature upon selective excitation of chlorophylls b at 640 or 650 nm. The kinetic model indicates a bidirectional excitation transfer over all CP29 chlorophylls a species, which is particularly rapid between the pure sites A1-A2 and A4-A5. Chlorophylls b in mixed sites act mostly as energy donors for chlorophylls a, whereas site B5 shows high and bidirectional coupling independent of the pigment hosted.     

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183-186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, 'blebs'.     

Linear dichroism of pigments associated with spherical chromatophores. Models of orientation in polyacrylamide gels

Linear dichroism and orientation of pigments in chromatophores of photosynthetic bacteria Chromatium minutissimum and Rhodospirillum rubrum using a novel method of orientation in polyacrylamide gel was studied. A model is proposed for orientation of spherical membranes of chromatophores or other similar vesicules. The value of linear dichroism is derived for known deformation of the gel and a certain angle between the transition dipole and a unit vector perpendicular to the membrane plane. The analysis of linear dichroism spectra permits calculation of angles between the normal to the membrane and the transition dipoles in Chr. minutissimum 65 degrees +/- 1.5 degrees (890 nm absorption band), 63 degrees +/- 1 degree (860 nm), 63 degrees +/- 1 degree (800 nm), 45.5 degrees +/- 1 degree (590 nm), 50.5 degrees +/- 0.5 degree (450--550 nm) and in Rsp. rubrum: 71 degrees +/- 1.5 degree (890 nm), 66.5 degrees +/- 1 degree (870 nm), 69 degrees +/- 1.5 degree (800 nm), 37 degrees +/- 0.5 degree (590 nm), 49.5 degrees +/- 0.5 degree (450--550 nm). The 860 nm band shift to shorter wave-lengths observed in Chr. minutissimum chromatophores treated with 0.01 M potassium ferricyanide is not related to reorientation of transition dipoles, but rather to certain changes of lipid-protein environment.     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Observation of anomalous carotenoid and blind chlorophyll activations in photosystem I under synthetic membrane confinements

The role of natural thylakoid membrane confinements in architecting the robust structural and electrochemical properties of PSI is not fully understood. Most PSI studies till date extract the proteins from their natural confinements that can lead to non-native conformations. Recently our group had successfully reconstituted PSI in synthetic lipid membranes using detergent-mediated liposome solubilizations. In this study, we investigate the alterations in chlorophylls and carotenoids interactions and reorganization in PSI based on spectral property changes induced by its confinement in anionic DPhPG and zwitterionic DPhPC phospholipid membranes. To this end, we employ a combination of absorption, fluorescence, and circular dichroism (CD) spectroscopic measurements. Our results indicate unique activation and alteration of photoresponses from the PSI carotenoid (Car) bands in PSI-DPhPG proteoliposomes that can tune the Excitation Energy Transfer (EET), otherwise absent in PSI at non-native environments. Specifically, we observe broadband light harvesting via enhanced absorption in the otherwise non-absorptive green region (500–580 nm) of the Chlorophylls (Chl) along with ~64% increase in the full-width half maximum of the Qy band (650–720 nm). The CD results indicate enhanced Chl-Chl and Chl-Car interactions along with conformational changes in protein secondary structures. Such distinct changes in the Car and Chl bands are not observed in PSI confined in DPhPC. The fundamental insights into membrane microenvironments tailoring PSI subunits reorganization and interactions provide novel strategies for tuning photoexcitation processes and rational designing of biotic-abiotic interfaces in PSI-based photoelectrochemical energy conversion systems.     

Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803

Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.     

Orientation of chlorophyll transition moments in the higher-plant light-harvesting complex CP29.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the F?rster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

Energy selection is not correlated in the Qx and Qy bands of a Mg-porphyrin embedded in a protein

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.     

Orientation of Photosystem-I pigments: low temperature linear dichroism spectroscopy of a highly-enriched P700 particle isolated from spinach

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The Q_Y transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700⁺ and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A₀, are all preferentially oriented perpendicular to the gel squeezing direction. The Q_Y transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.Abbreviations PSI Photosystem I - P700 primary electron donor of PSI - A₀ primary electron acceptor of PSI     

Orientation of the Chromophore Plane in Purple Membrane

Excitation of bacteriorhodopsin (BR) in its β absorption band drives a photocycle identical in the millisecond range, to that excited in the α band of BR. The relative contribution of the two transition dipoles distinguished in the β band to the initiation of the photocycle was established by photoselection experiments. Having this information the orientation of the chromophoric plane was specified by electric dichroism measurements.     

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q(Y) dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9 degrees at 677 nm, 20 degrees at 670 nm and 26 degrees at 662 nm); the Q(Y) dipole of Chl c was found to be oriented at 29 degrees with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q(Y) dipoles of Chl b to lie preferentially in the plane of the membrane (23 degrees at 653 nm and 30 degrees at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.     

Orientation and linear dichroism characteristics of porphyrin-DNA complexes

The linear dichroism spectra of complexes of tetrakis(N-methyl-4-pyridinio)prophine (H2TMpyP) and its zinc(II) derivative (ZnTMpyP) with DNA oriented in a flow gradient have been investigated. The dichroism of H2TMpyP determined within the Soret band and the Qy band system is consistent with an intercalative conformation in which the plane of the porphyrin ring system is nearly parallel to the planes of the DNA bases. In the case of ZnTMpyP on the other hand, the porphyrin ring system is inclined at angles of 62-67 degrees with respect to the axis of the DNA helix. The pyridyl groups in both cases are characterized by a low degree of orientation with respect to the axis of the helix. In contrast to H2TMpyP which does not significantly affect the degree of alignment of the DNA in the flow gradient, the binding of ZnTMpyP causes a significant decrease (about 50% for a base pair/ZnTMpyP ratio of 20) in the intrinsic dichroism at 260 nm due to the oriented DNA bases; the binding of ZnTMpyP to DNA either gives rise to regions of higher flexibility or causes bends or kinks at the binding sites. Increasing the ionic strength has little influence on the linear dichroism of the ZnTMpyP-DNA complexes, but the number of molecules bound at intercalation sites diminishes in the case of the H2TMpyP-DNA complexes; the accompanying changes in the linear dichroism characteristics suggest that external H2TMpyP complexes are formed at the expense of intercalation complexes.(ABSTRACT TRUNCATED AT 250 WORDS)