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Infrared and visible spectroscopy is used to investigate the primary processes in bacterial photosynthesis. Experiments are presented showing convincingly that the first electron transfer step in primary photosynthesis leads to the radical pair state P+BA- where the electron has reached the accessory bacteriochlorophyll BA of the active branch. Ultrafast spectroscopy in the mid infrared indicates that the electron transfer to the bacteriochlorophyll is preceded by a very fast (200 fs) reaction in the excited electronic state of the special pair.

A novel mode to apply photosensitizing drugs specifically to tumor tissue using the principles of polyphasic tumor therapies is outlined. Key compounds are tumor specific functionalized antibodies with reduced immunogenicity. These bind to drug inclusion complexes in a multiplicative manner. Drug inclusion complexes are designed on the basis of tethered functionalized (beta) -cyclodextrin dimers with maximum affinity to porphyrinoid photosensitizers forming monomeric chlathrates. To enhance porphyrin-cyclodextrin interaction peripheral groups of the porphyrin have to be chemically modified. The development of the method is not yet completed. First results are demonstrated.

Publisher Summary This chapter focuses on the diode array detection in liquid chromatography. In a diode array detector, the spectral information is measured simultaneously at many different wavelengths and at repetition rates that allow the collection of several spectra during the elution of a single chromatographic peak. This spectral information provides a safer means of identification, but it also has other advantages. First, although single-wavelength detectors are inherently more sensitive, this sensitivity often cannot be used fully because the different components in a mixture may have rather different absorption (or fluorescence) properties which have to be compromised. Because the entire spectral information can be used in a diode array detector, the wavelength of highest sensitivity can be chosen for each component separately. Second, overlapping bands can be analyzed much more reliably by chemometric methods as long as the components involved differ in their absorption or fluorescence.

The decay of the surviving cell fraction, F, during photodynamic treatment of a cell population, F0, vs. energy dose obeys an exponential law. The integrated logarithmic form yields a straight line with intercept 0 and slope -k. The reciprocal slope yields LD90, i.e., the energy fluence (J/cm2) necessary to kill 90% of the cell population F0. In reality there are deviations from this simple set of equations. At low power densities most photosensitizers are unable to kill cells. This leads to an `optical threshold' without cell destruction but frequently with cell stimulating effects, so called `overshots.' This general phenomenon occurs in a very pronounced form with melanotic melanoma cells.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H218O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-71-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H218O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 131-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599–602]. The incorporation of isotope led to as much as 77% enrichment of the 131-oxo group of chlorophyll a: assuming identical incorporation into the 131 oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The high enrichment using 18O2, and the absence of labelling by H218O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.

The primary electron transfer in reaction centers of Rhodobacter sphaeroides is studied by subpicosecond absorption spectroscopy with polarized light in the spectral range of 920-1040 nm. Here the bacteriochlorophyll anion radical has an absorption band while the other pigments of the reaction center have vanishing ground-state absorption. The transient absorption data exhibit a pronounced 0.9-ps kinetic component which shows a strong dichroism. Evaluation of the data yields an angle between the transition moments of the special pair and the species related with the 0.9-ps kinetic component of 26 +/- 8 degrees. This angle compares favorably with the value of 29 degrees expected for the reduced accessory bacteriochlorophyll. Extensive transient absorbance data are fully consistent with a stepwise electron transfer via the accessory bacteriochlorophyll.

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of 18O2. This suggests that a mono-oxygenase is involved in the oxidation of its methyl group precursor. The concomitant enrichment of about 75% of the 131-oxygen confirms the well-documented finding that this oxo group, in both chlorophyll a and b, also arises from O2. High 18O enrichment into the 7-formyl oxygen relative to the substrate 18O2 was achieved by optimization of the greening conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen.

Triplet state electron paramagnetic resonance (EPR) experiments have been carried out at X-band on Rb. sphaeroides R-26 reaction centers that have been reconstituted with the carotenoid, spheroidene, and exchanged with 13(2)-OH-Zn-bacteriochlorophyll a and [3-vinyl]-13(2)-OH-bacteriochlorophyll a at the monomeric, 'accessory' bacteriochlorophyll sites BA,B or with pheophytin a at the bacteriopheophytin sites HA,B. The primary donor and carotenoid triplet state EPR signals in the temperature range 95-150 K are compared and contrasted with those from native Rb. sphaeroides wild type and Rb. sphaeroides R-26 reaction centers reconstituted with spheroidene. The temperature dependencies of the EPR signals are strikingly different for the various samples. The data prove that triplet energy transfer from the primary donor to the carotenoid is mediated by the monomeric, BChlB molecule. Furthermore, the data show that triplet energy transfer from the primary donor to the carotenoid is an activated process, the efficiency of which correlates with the estimated triplet state energies of the modified pigments.

During rapid extraction of chlorophylls from maize leaves under reducing conditions with methanol containing NaBH4, chlorophyll a remained unchanged but chlorophyll b yielded [7-hydroxymethyl]-chlorophyll b. The 3-vinyl group of chlorophyll b was also reduced forming significant am ounts, up to 60%, of [3-ethyl]-[7-hydroxymethyl]-chlorophyll b. This was unexpected since this reduction of the 3-vinyl group does not occur when isolated chlorophyll b is treated in an identical manner with methanolic borohydride. The vinyl-group of chlorophyll a is not reduced during the same extraction conditions suggesting that the presence of a formyl or hydroxyethyl group at C-7 is necessary. The presence of 8-hydroxyquinoline and NaBH4 in equimolar (16.5 m M) concentrations strongly inhibits the reduction of the 3-vinyl group of chlorophyll b in leaf extracts

Kinetic and equilibrium features of the epimerization in six C132-epimer pairs of chlorophyllous pigments, namely chlorophyll (Chl) a/a′, Chl b/b′, pheophytin (Pheo) a/a′, Pheo b/b′, C20-chlorinated Chl (Cl–Chl) a/a′, and Cl–Pheo a/a′, catalyzed by bases (triethylamine, pyrrolidine, and piperidine) and by Lewis acids (magnesium acetylacetonate and magnesium chloride), have been studied in diethyl ether and methanol. A significant difference was noted in the epimerization rate and equilibrium epimeric composition among the pigment systems. The difference in the rate constants was rationalized in terms of the ease in C132-proton detachment, affected by the ring π-electron density which in turn depends on the electron donation or withdrawal by the central metal and/or ring substituents. For Lewis acid-catalysis, a dominant factor appears to be the stabilization of the C131-enolate anion via formation of a loose complex with extraneous Mg2+. A synergistic action of a base (triethylamine) and a Lewis acid (Mg2...

The exchangeability of the bacteriopheophytins at sites HA and HB with modified (bacterio)pheophytins (=(B)Phe)*) was tested in reaction centers (RC) of Rhodobacter spheroides R26. An exchange at both sites is possible with Pyro-BPhe a lacking the 132-COOCH3 group, and with three plant-type pheophytins: Phe a (which contains a 3-vinyl-group), 132-hydroxy-Phe a (which contains in addition a hydroxy group), and [3-acetyl]-Phe a (which differs from BPhe a only by the unsaturated ring II). In all cases, the exchange appears to be easier at the HB -site. An exchange only at this site, was obtained with BPhe a gg in which the esterifying phytol is replaced by geranyl-geraniol, and with 132-hydroxy-Phe a. Environment-induced red-shifts (EIRS) are observed with all pigments, and they are in the range of the ones known for the native BPhe a. Strong optical activity is induced in most pigments. Shifts in the absorptions of the monomeric BChls at sites BA,B indicate an interaction with the BPhes sites HA,B or an indirect structural effect.

Butenandtstrasse 11, D-81377 Munich, Germany Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA Department of Biology and Biotechnology, Ruhr-University Bochum, 44780 Bochum, Germany Department of Biology I–Botany, Ludwig-Maximilian-University, D-80638 Munich, Germany Department of Physics and Astronomy, Ohio University, Athens, Ohio 45701, USA Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia

Biliproteins are widespread pigments in nature, and perform a variety of rather different functions (Kayser, 1985; Scheer, 1982; Braslaysky et al., 1983; Ruediger and Scheer, 1983): The phycobiliproteins occurring in cyanobacteria, rhodophytes and cryptophytes, function as photosynthetic light-harvesting pigments. Phytochromes are sensory photoreceptors in plants and algae, and the putative adaptochromes and photomorphochromes of cyanobacteria are probably also biliproteins. The bilirubinserum albumin conjugates are involved in transporting this poorly water-soluble pigment, and many invertebrates contain biliproteins which play a role in their pigmentation, but may have additional functions.

Fourier-transform infrared difference spectra between the red-absorbing and far-red-absorbing forms of oat phytochrome have been measured in H2O and 2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)- and (5Z,10Z,15E)-isomers of 2,3,7,8,12,13,17,18-octaethyl-bilindion (Et8-bilindion), 2,3-dihydro-2,3,7,8,12,13,17,18-octaethyl-bilindion (H2Et8-bilindion), and protonated H2Et8-bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8-bilindion and H2Et8-bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The 2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red-absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far-red-absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure.

Fluorescence and initiation of photoreactions are problems frequently encountered with resonance Raman spectroscopy of photobiological systems. These problems can be circumvented with Fourier-transform Raman spectroscopy by using the 1064-nm wavelength of a continuous wave neodymium-yttrium/aluminum-garnet laser as the probing beam. This wavelength is far from the absorption band of most pigments. Yet, the spectra of the investigated systems--bacteriorhodopsin, rhodopsin, and phycocyanin--show that these systems are still dominated by the chromophore, or that preresonant Raman scattering is still prevalent. Only for rhodopsin were contributions of the protein and the membrane discernible. The spectra of phycocyanin differ considerably from those obtained by excitation into the UV-absorption band. The results show the usefulness of this method and its wide applicability. In addition, analysis of the relative preresonant scattering cross sections may provide a detailed insight into the scattering mechanism.

The primary reaction of bacterial photosynthesis — an electron transfer via several prosthetic groups in the so-called reaction center — proceeds extremely rapid on the time-scale of picoseconds. A series of recent experiments gave the following picture of this charge transfer process (data taken for reaction centers from Rhodobacter (Rb.) sphae-roides /1–4/): After excitation of the lowest excited singlet state of the primary electron donor (a “special pair” of bacteriochlorophyll molecules) the excited electronic state P* lives for approximately 3.5ps. The decay of P* is related with the electron transfer away from P. From several time-resolved experiments it was concluded that this first charge transfer carries the electron directly to the bacteriopheophytin H /1,2/. Only very recently we could demonstrate the existence of an additional short-lived intermediate prior to the reduction of the bacteriopheophytin H /4/. We interpreted this intermediate as P+B‒, i.e. the state where the electron from the special pair P has reduced the monomeric bacteriochlorophyll B to the anion radical B‒. In the final picosecond reaction the electron arrives (with a time constant of 200 ps) at the quinone Qa. It is the purpose of this paper to present additional experimental data supporting a sequential electron transfer via the accessory bacteriochlorophyll.

Photosynthetic conversion of light into chemical energy starts via a series of electron transfer reactions in pigment-protein complexes called reaction centers (RC’s). The most direct access to the primary reaction dynamics offers time resolved optical spectroscopy. During the past few years, this technique has been continuously improved permitting advanced experiments with high temporal and amplitude resolution. In this paper, we show, that RC’s from Rhodobacter (Rb.) sphaeroides and Rhodopseudomonas (Rps.) viridis exhibit common features in the absorption transients. This points to a substantial similarity of the elementary molecular processes. This fact is not self-evident, since different polypeptides and different types of bacteriochlorophylls (BChl) and bacteriopheophytins (BPh) are present in various reaction centers, e.g. BChl a and BPh a are essential pigments in the reaction centers of Rb. sphaeroides while BChl b and BPh b are active in the RC’s of Rps. viridis. For both reaction centers x-ray structures are now available /1–3/. It was shown that the prosthetic groups and neighbouring amino acids are in a very similar arrangement: most importantly are two BChl molecules in close contact which act as the primary electron donor P. The other pigments are arranged in two branches, A and B. Starting from the primary donor, the special pair P, one finds a monomeric bacteriochlorophyll (B), a bacteriopheophytin (H), and a quinone (Q) on each branch. It was shown that the electron transfer occurs via the A-branch and that after about 3 – 4 ps a radical pair P+HA − is formed. Approximately 200 ps later the electron reaches the quinone QA building the intermediate P+QA −.

Chromophores of C-phycocyanin and phycoerythrο-cyanin have been chemically modified by reduction to rubins , bleaching , photoisomerization , or perturbation with bulky substituents. Pigments containing modified chromophores, or hybrids containing modified and unmodified chromophores in individual protomers have been prepared. All modifications inhibit the association of the (aß)-protomers of these pigments to higher aggregates. The results demonstrate a pronounced effect of the state of the chromophores on biliprotein quaternary structure. It may be important in phycobi1isome assembly , and also in the dual function of biliproteins as (i) antenna pigments for photosynthesis and (ii) reaction centers for photomor-phogenesis.

Phycocyanin plays an important role in collecting light energy and transfering it to the reaction center (1,2). In vivo, this chromoprotein is highly aggregated as part of the phycobilisomes (3–5). In vitro, in buffers of low ionic strenght, the phycobilisomes dissociate. Under these conditions, phycocyanin of Mastigocladus laminosus is aggregated to ring-shaped trimers (αβ)3. The α-subunit carries one (α-84) the β-subunit two covalently linked cyanobilin-chromophores (β-84 and β-155) (6,7). These trimers are generally associated with linker-peptides, which can principally function as structural elements, as “terminators” of antenna rod building, but are also involved in fine tuning of the chromophore spectra for optimum energy transfer (3–6). In order to investigate the role of individual chromophores in energy transfer, we are modifying distinct chromophores within energy transfering chromophore ensembles. During such studies, we have now found that chromophore modifications have also a profound influence on protein-chromophore interactions and chromoprotein aggregation. It is this aspect which is presented here.

The past five years after solving the crystal structure of the bacterial reaction center from Rhodopseudomonas (Rp.) viridis [1] — and the first Feldafing meeting [2] — have witnessed a remarkable concentration of interdisciplinary research on this pigment-protein complex. The importance of the reaction center is related to its key function in transforming photons into electrochemical energy and, in more general terms, to the fact, that it is the first membrane protein of which the three-dimensional structure is known. Beyond these central biological bearings, the reaction center has a stimulating effect on many developments in related fields. Its complex pigment-protein and pigment-pigment interactions required refinement of existing and development of novelspectroscopic techniques. Since the reaction center constitutes the best-defined supramolecular electron donor/acceptor system in chemistry, it has the potential of becoming a test case for electron transfer theories. This year’s Feldafing II conference intended to provide a status-report on the present understanding of reaction center function, highlighting the new biochemical, spectroscopic and theoretical approaches. Still under the impression of the conference we will try to give our personal views and some leading references*, preferentially to papers within this book, on the spectacular progress and limitations related to biochemical and genetic modifications of the reaction center collected in Part III.

Chlorophyll (Chl) derivatives are readily and regiospecifically chlorinated at C-20, the free methine-bridge next to the reduced ring D (1–3). The interest in chlorinated chlorophylls has recently been renewed after their isolation from extracts of various green plants, algae and cyanobacteria (4–9) and a suggested relation to photosystem I of oxygenic photosynthesis (4, 5). This relation, and hence the involvement in photosynthesis, has been met with criticism and now been disproved (8, 10–12).

The authors show that energy transfer is a feasible mechanism for exciting fluorescence of single light-harvesting complexes with different characters of the fluorescing state. This approach is applied for excitons consisting of 18 strongly coupled bacteriochlorophylls in light-harvesting complex 2 as well as for peridinin-chlorophyll-protein monomers containing either two chlorophyll a molecules or a pair of chlorophyll a and chlorophyll b characterized with the emission energy difference of 400cm−1. Using this method the authors are able to observe fluorescence spectral dynamics on the scale comparable or larger than the emission linewidth of a single chromophore.

The photostabilities of bacteriochlorophyll a and several of its derivatives, which are of interest as potential sensitizers in photodynamic tumor therapy, were investigated. The pigments were irradiated with light >630 nm in organic solvents (acetone, tetrahydrofuran, pyridine, methanol, ethanol, n-propanol, 2-propanol and toluene) and in aqueous detergent solutions (cetyl-trimethyl-ammonium bromide [CTAB], lauryldimethyl-aminoxide [LDAO] or sodium dodecyl-sulfate [SDS] and Triton X-100 [TX100]). Their stabilities in these different solvents were determined in the presence and absence of an external sensitizer (pyromethyl-pheophorbide a), oxygen, sodium ascorbate and inert gas (Ar) or vacuum. The photodegradation products of bacteriochlorophyll a in acetone solution were isolated, purified by HPLC and analyzed by their absorption spectra and mass spectroscopy. Besides the well-known dehydrogenation products, such as [3-acetyl]-chlorophyll a, which were obtained as by-products, the major products had low absorption in the visible-near infrared spectral range. The spectral signature of the major component of these products was characteristic of linear open-chain tetrapyrroles, but they lacked the characteristic protonation-deprotonation behavior and reactivity of bilins with Zn(++).

Bacteriochlorophyll (BChl) derivatives (with central Mg replaced by metal "M") ([M]-BChl with M = 2H, Mg, Zn, Pd, Cu) have been investigated for their photodynamic capacity and stability toward photodegradation in organic solvents and aqueous micellar solution. A protocol has been developed for screening new sensitizers. BChl and [Zn]-BChl are efficient sensitizers, but they are also quickly degraded by the reactive oxygen species (ROS) produced by autosensitization, as well as by hetero-sensitization with 17(4)-methyl-13(2)-demethoxycarbonyl-pheophorbide a (MPP). Photostable [Cu]-BChl is a poor sensitizer, whereas [Pd]-BChl and bacteriopheophytin a are not only very efficient sensitizers but are also very stable toward ROS. beta-Carotene is no efficient physical quencher of ROS in the system; rather, it acts as a photochemical quencher that competes with [M]-BChl and undergoes photooxygenation at high rates. Photolability seems to depend on the pigment oxidation potential and, in parallel, on the presence of central metals preferring coordination numbers higher than 4, whereas photodynamic capacity depends on long excited state life-times of the pigment or efficient intersystem crossing (or both).

Carotenoids are well-known physical quenchers of chlorophyll excited states and reactive oxygen species both in vivo and in vitro. They may also be involved in chemical quenching undergoing, e.g. isomerizations or oxidations. We have found that b-carotene (Car) in aerobic acetone is rapidly oxygenated under strong illumination with red light (lexc $ 630 nm) in the presence of bacteriopheophytin a. At the same time the photosensitizer undergoes only slight (,10%) photodegradation. By preparative high-performance liquid chromatography as many as seven major products of oxygen attachment to Car have been isolated. Their molecular masses show that Car sequentially accumulates up to six oxygen atoms while its C 40 -skeleton remains intact.

The reaction of biliproteins with sodium dithionite has been studied. The reagent is selective towards the chromophores. In denatured phycocyanin from Spirulina platensis, all three chromophores react to form yellow “phycorubin”, whereas only 1/3 of the chromophores react in native phycocyanin in a non-statistical manner. From reversion experiments, it can be shown, that the thermodynamic stability of the chromophores towards reaction with dithionite is increased in the native pigment. Similarly, native phytochrome in its Pr form reacts only partially to a pigment absorbing at both 420 and 660 nm. The same product is formed from native Pfr, indicating both a reversion to Pr and a partial reduction.

The influence of aggregation and temperature on the excited state kinetics of C-phycocyanin from Mastiqocladus laminosus has been studied. Polarized fluorescence decay curves have been recorded using a synchronously pumped dye laser in conjunction with a synchroscan streak camera. The experimental data for all samples can be fit satisfactorily assuming a biexponential decay law. Fluorescence depolarization times have been interpreted in terms of energy transfer among the different chromophores. The influence of temperature is only moderate on the intra­molecular relaxation, but pronounced on the rates of energy transfer. Both are dependent on the size of the aggregate. The biexponential decay of the α-subunit containing only one chromophore, indicates the presence of different subsets of chromophores in these samples. The results are discussed in terms of variations of the chromophore arrangements upon temperature induced changes in the protein conformation.

The reaction of bilirubins with aromatic diazonium salts in alcoholic solvents leads to an equimolar mixture of two types of products. One is the well-known 9-azopyrromethenone. The other is a yellow product (λmax ≈ 420 nm) identified as 9-alkoxymethylpyrromethenone, the alkoxy-substituent being derived from the solvent. Thus, reaction of the symmetrically substituted bilirubins IIIα (1c) and XIIIα (1b) in methanol with diazotized sulfanilic acid yields one mole of the azopigments (4b and 4a), respectively, and one mole of the corresponding 9-methoxymethylpyrromethenones (2b and 2a). Bilirubin IXα (1a) consequently yields a mixture of all four products. The two resulting 9-methoxymethylpyrromethenones were separated by chromatography and identified as 2a and 2b. They can react further with the diazonium salts to give the corresponding 9-azopyrromethenones, but the reaction is much slower than that of bilirubin, which explains the observed product distribution. These results are discussed in relation to earlier work.

We have shown that both the native C-phycocyanin and its corresponding free biline chromophore undergo reversible, low-temperature photochemistry. We attribute this photochemistry to reversible proton-transfer processes and utilize the observed photoreaction for photochemical hole burning (PHB). Using narrow-band PHB experiments, we have been able to perform high-resolution optical studies and show that the protein-chromophore assembly forms a very rigid structure. The results lead to the conclusion that the light-induced proton transfer occurs most probably in the triplet state.

The circulardichroism spectra (cd) of native and (partially) urea-denaturated C-phycocyanin (PC) in the spectral range 700-210 nm are presented. The large ellipticities observed in the chromophoric region of native PC are retained in the fully denatured state (8 ᴍ urea). This is similar to the behavior of the red form of phytochrome (Pr), but in contrast to C-phycoerythrin (PE). These differences are rationalized in terms of epimeric equilibria between P- and M-helix shaped chromophores. Depending on the number and location of the chirality centers present in the tetrapyrrol moities a priori, the excess populations of the inherently chiral P- and M-helices differ, thus accounting for large (PC, Pr) or small (PE) ellipticities in the denatured pigments. Hence, the large optical activity observed for the former is generated by an excess population of the P-helix induced by the asymmetric C-2, C-3 and C-3'. In PE the additional chirality center at C-16 counteracts the influence of the others. The excess population of the inherently chiral species is therefore lowered, in agreement with the nearly vanishing cd reported for denatured PE. The cd has also been studied at intermediate urea concentrations. Unfolding of PC with urea can be interpreted from these data as a stepwise process. Monitoring the urea induced unfolding of PC by cd at different wavelengths (λ = 220, 345, and 610 nm); the “melting point” of the apoprotein (4 -5 ᴍ urea) coincides with the extrema of the titration curves obtained in the chromophore region (345, 610 nm). These results give direct evidence for the existence of an intermediate species whose population reaches a maximum at 4-5 ᴍ urea.

C-phycocyanin from the cyanobacterium, Mastigocladus laminosus, and its subunits have been treated with ρ-chloromercuribenzenesulfonate (PCMS). A single reactive site was found on the 13- subunit, and assigned to the single free cystein-β109. The concomitant spectral changes (absorp­tion, fluorescence, circular dichroism), together with the known close proximity of cys-β109 to chromophore β82, allowed an unambiguous assignment of the three spectrally, biochemically and functionally different chromophores to specific binding sites on the two peptide chains (α84: 616-618, β82: 622-624, β153: 598-600 nm).

Highly frequency selective photochemistry at cryogenic temperatures is used to gain information on the excited state dynamics of large biomolecular aggregates, the phycobilisomes from the blue–green alga (cyanobacterium) Masticogladus laminosus. In particular, we show that in spite of the well organized structure of these aggregates disorder on a microscopic level dominates the optical spectra. The hole burning reaction in the resonantly excited chromophores is most probably due to a conformational change in the neighborhood of the chromophore. From the widths of the holes energy transfer times between different pigments on the order of 16 ps are determined. These transfer times are independent of the excitation energy.

The chromophore-protein interactions of C-phycocyanin (C-PC) from Spirulina platensis have been studied by following the partial and complete denaturation with UV-Vis spectroscopy. From comparison with published MO calculations, an elongated conformation of the chromophore is suggested for native C-PC, a cyclic one for denatured C-PC. By means of partial denaturation, a step­ wise unfolding of the protein has been demonstrated. The presence of at least two sets of spectro­ scopically different diromophores is suggested from the partial denaturation and low temperature experiments.

The reaction of several plant chlorophyll-protein complexes with NaBH, has been studied by absorption spectroscopy. In all the complexes studied, chlorophyll b is more reactive than Chl a, due to preferential reaction of its formyl substituent at C-7. The complexes also show large variations in reactivity towards NaBH, and the order of reactivity is: LHCI > PSI1 complex > LHCII > PSI > P700 (investigated as a component of PSI). Differential pools of the same type of chlorophyll have been observed in several complexes. Parallel work was undertaken on the reactivity of micellar complexes of chlorophyll a and of chlorophyll b with NaBH, to study the effect of aggregation state on this reactivity. In these complexes, both chlorophyll a and b show large variations in reactivity in the order monomer > oligomer > polymer with chlorophyll b generally being more reactive than chlorophyll a. It is concluded that aggregation decreases the reactivity of chlorophylls towards NaBH, in vitro, and may similarly decrease reactivity in naturally-occurring chlorophyll-protein complexes.

An electron spin echo modulation frequency analysis of P+-860 is performed in 14N chromatophores of Rhodospirillum rubrum, and in 15N substituted reaction centers from Rhodopseudomonas sphaeroides 2.4.1. For the 14N material two sets of nuclear quadrupole parameters are obtained. From the frequencies found for 15N reaction centers the perpendicular, parallel and isotropic hyperfine coupling constants of three of the four bacteriochlorophyll nitrogen nuclei are inferred.

Complex mixtures of pheophytins esterified with different C-20 diterpene alcohols have been separated by high-performance liquid chromatography in a reversed-phase system. The isomers of some monomeric and dimeric biliverdins have also been studied. Analytical and semi-preparative work is reported.