Julio C. de Paula

Depolarized Resonance Light Scattering by Porphyrin and Chlorophyll a Aggregates

A quantum mechanical model is developed for the observed resonance enhancement of light scattering by aggregates of electronically interacting chromophores. Aggregate size, monomer oscillator strength, extent of electronic coupling, and aggregate geometry are all important determinants of intensity in resonance light scattering (RLS) spectra. The theory also predicts the value of the depolarization ratio (rho(v)(90)) of RLS for a given aggregate geometry. These results are used to interpret the RLS depolarization ratios of four aggregates: tetrakis(4-sulfonatophenyl)porphine aggregated at low pH (rho(v)(90) = 0.17 at 488 nm), trans-bis(N-methylpyridinium-4-yl)-diphenylporphinato copper(II) aggregated in 0.2 M NaCl solution (rho(v)(90) = 0.13 at 450 nm) and on calf thymus DNA (rho(v)(90) = 0.20 at 454 nm), and chlorophyll a aggregates in formamide/water (rho(v)(90) = 0.23 and 0.32 at 469 and 699 nm, respectively). The analysis is consistent with a J-aggregate geometry for all four systems. Furthermore, the specific values of rho(v)(90) allow us to estimate the orientation of the monomer transition dipoles with respect to the long axis of the aggregate. We conclude that depolarized resonance light scattering spectroscopy is a powerful probe of the geometric and electronic structures of extended aggregates of strong chromophores.

Interactions of copper(II) porphyrins with DNA

Abstract The interactions of three cationic water soluble copper(II) porphyrins, differing in peripheral substituents, with calf thymus DNA are described. Tetrakis(N-methylpyridinium-4-yl)porphinecopper(II) behaves as a simple intercalator under the conditions investigated, whereas tetrakis(4-N,N′,N″-trimethylanilinium)porphinecopper(II), binds externally, with some limited aggregation under high drug load conditions. In contrast, trans-bis(N-methylpyridinium-4-yl)diphenylporphinecopper(II) (t-CuPagg), like the free-base t-H2Pagg from which it is derived, is capable of forming extended electronically coupled arrays while bound to the DNA template. These arrays have been investigated using a combination of extinction spectroscopy, circular dichroism, RLS and resonance Raman spectroscopy. They are found to contain 105–106 porphyrin units, arranged in long, narrow organized structures. The kinetics of assembly of t-CuPagg is reported on three DNAs: ct DNA, poly(dG–dC)2 and poly(dA–dT)2. A non-conventional autocatalytic model first proposed for t-H2Pagg assembly formation is successful at fitting these data, permitting direct comparisons of kinetic parameters for the two porphyrins. It is found that the catalytic rate constant (kc) is considerably smaller for t-CuPagg than for t-H2Pagg under comparable conditions, and that the template rigidity fosters assembly formation. We also report the resonance Raman spectra of t-H2Pagg/ct DNA and t-CuPagg/ct DNA complexes. Aggregation on the DNA template changes the intensity pattern of the porphyrins resonance Raman spectra, with some low- and high-frequency bands becoming strongly enhanced upon aggregation. We conclude that aggregation-enhanced resonance Raman spectroscopy is a useful probe of aggregation in porphyrin–DNA complexes that also gives detailed information about structural changes that accompany the aggregation process.

The Use of Cyanobacteria in the Study of the Structure and Function of Photosystem II

Oxygenic photosynthesis occurs in plants, green algae, and procaryotic cyanobacteria. Two chlorophyll-containing photosystems cooperate to transfer electrons from water to NADP+. Photosystem II is the membrane protein complex that carries out the light-catalyzed oxidation of water and reduction of plastoquinone. The reaction center is composed of both intrinsic and extrinsic proteins; the prosthetic groups involved in electron transfer include chlorophyll, pheophytin, quinone, tyrosine residues, and a manganese cluster. Cyanobacteria have emerged as a convenient system with which to study the structure and function of Photosystem II for two reasons. Firstly, isotopic labeling experiments are possible in this organism, facilitating many types of biophysical experiments. Secondly, site-directed mutagenesis is easily performed. This chapter will review what is known about the structure and function of Photosystem II with particular emphasis on the use of cyanobacteria in such studies. Areas in which there are significant differences between plants and cyanobacteria will be highlighted.

Self Assembly of Coiled-Coil Peptide−Porphyrin Complexes

We are interested in the controlled assembly of photoelectronic materials using peptides as scaffolds and porphyrins as the conducting material. We describe the integration of a peptide-based polymer strategy with the ability of designed basic peptides to bind anionic porphyrins in order to create regulated photoelectronically active biomaterials. We have described our peptide system in earlier work, which demonstrates the ability of a peptide to form filamentous materials made up of self-assembling coiled-coil structures. We have modified this peptide system to include lysine residues appropriately positioned to specifically bind meso-tetrakis(4-sulfonatophenyl)porphine (TPPS(4)), a porphyrin that contains four negatively charged sulfonate groups at neutral pH. We measure the binding of TPPS(4) to our peptide using UV--visible and fluorescence spectroscopies to follow the porphyrin signature. We determine the concomitant acquisition of helical secondary structure in the peptide upon TPPS(4) binding using circular dichroism spectropolarimetry. This binding fosters polymerization of the peptide, as shown by absorbance extinction effects in the peptide CD spectra. The morphologies of the peptide/porphyrin complexes, as imaged by atomic force microscopy, are consistent with the coiled-coil polymers that we had characterized earlier, except that the heights are slightly higher, consistent with porphyrin binding. Evidence for exciton coupling in the copolymers is shown by red-shifting in the UV--visible data, however, the coupling is weak based on a lack of fluorescence quenching in fluorescence experiments.

Formation of the S2 state and structure of the Mn complex in photosystem II lacking the extrinsic 33 kilodalton polypeptide

Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487–6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca2+. The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.

Heme A and A3 environments of plant cytochrome C oxidase

The structures of hemes a and a3 of maize and wheat germ cytochrome c oxidase were investigated by resonance Raman spectroscopy. Comparison between the plant and mammalian cytochrome oxidases revealed that (i) the vinyl groups associated with hemes a and a3 vibrate at higher frequencies in the plant enzyme than in the mammalian enzyme, suggesting different degrees of interaction between the heme cores and their periphery; (ii) aside from the geometry of the vinyl group, the structure of heme a3 in plant cytochrome oxidase is essentially unchanged from that of its mammalian counterpart; (iii) the vibrational band associated with the formyl group of reduced heme a shows relatively weak enhancement in the Soret-excited resonance Raman spectra of maize and wheat germ cytochrome oxidase, suggesting that the formyl group of ferrous heme a in the plant enzymes is conjugated only slightly to the porphyrin ring; and (iv) for oxidized heme a, the formyl vibration is strongly enhanced, but its frequency indicates a weaker interaction with the protein milieu relative to the mammalian enzyme. These observations suggest that the local environment around the formyl position of the heme a chromophore differs in the plant and mammalian cytochrome oxidases. The implication of the latter feature in the mechanism of proton pumping by cytochrome oxidase is discussed.

Chlorophyll-Protein Interactions in Photosystem II. Resonance Raman Spectroscopy of the D1-D2-cytochrome b559 Complex and the 47 kDa Protein

Recent advances in the biochemistry of plant photosystem II (PSII) have led to the proposal that the complex composed of the D1 (32 kDa), D2 (34 kDa), and cytochrome b559(4 and 9 kDa) polypeptides constitutes the reaction center core (1). This complex, though similar in many respects to the reaction center complex of purple bacteria, has unique characteristics. Chief among these is its pigment stoichiometry. Unlike the bacterial reaction center complex, which binds six chlorin molecules, the D1-D2-cytochrome b559 complex has as many as 13 chlorin molecules (2).

Structures and Organization on the Oxidizing Side of Photosystem II

Recently, there has been substantial progress in developing methods by which to resolve Photosystem II biochemically and to manipulate it genetically (for reviews, see 1–3 and references therein). The biochemical developments have allowed samples with significantly higher Photosystem II concentrations to be prepared. This has facilitated the application of a number of spectroscopic techniques and has allowed new physical techniques to be brought to bear on outstanding problems in PSII. The advances in genetic operations that can be applied to PSII proteins now extends to both core polypeptides, Dl and D2. As a result, important components in the electron transfer reactions that precede O2 evolution have been identified and the relevance of C2 symmetry arguments, in analogy to the bacterial reaction center, has been demonstrated (4). Although this analogy must eventually break down simply because of the different functions that occur in bacterial and PSII reaction centers, determining where the divergence occurs is of importance. Already, for example, there are indications that the pigment composition of the PSII reaction center is considerably more complex than that of the bacterial reaction center (5).