Structure of c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 A: structural implications for thermal stability in phycobilisome assembly

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has been determined by molecular replacement to 2.5 A resolution. The crystal belongs to space group R32 with cell parameters a=b=188.43 A, c=61.28 A, alpha=beta=90 degrees, gamma=120 degrees, with one (alphabeta) monomer in the asymmetric unit. The structure has been refined to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 A. The crystals were grown from phycocyanin (alphabeta)(3) trimers that form (alphabeta)(6) hexamers in the crystals, in a fashion similar to other phycocyanins. Comparison of the primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella diplsiphon and the thermophilic Mastigocladus laminosus were performed. We show that each level of assembly of oligomeric phycocyanin, which leads to the formation of the phycobilisome structure, can be stabilized in thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each association interface. In addition, a significant shift in the position of ring D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin, enables the formation of important polar interactions at both the (alphabeta) monomer and (alphabeta)(6) hexamer association interfaces.     

Use of immobilized light-harvesting chlorophyll a/b protein to study the stoichiometry of its self-association.

D. J. Davis & E. L. Gross (1976) Biochim. Biophys. Acta 449, 554-564 previously observed that the light-harvesting chlorophyll a/b protein or chlorophyll protein complex II self-associated as determined by ultracentrifugation. We have determined the stoichiometry of complex formation by immobilizing the monomer on ethylenediamine-Sepharose 4B and determing the ability of immobilized protein to bind the free protein. The amount of soluble protein bound to the immobilized protein increased as the concentration of soluble protein increased. The binding was maximal between pH 7 and 8. The maximum binding was three molecules bound per one molecule of protein immobilized. These results indicate that a tetramer is the intrinsic structural unit of the light-harvesting chlorophyll a/b protein in the chloroplast membrane. Upon complex formation, the chlorophyll fluorescence was decreased without any spectral change. The maximum binding was approximately doubled upon addition of 0.5 mM CaCl2 whereas 5 mM NaCl had no effect. Addition of CaCl2 had no effect on the fluorescence of the monomer. The light-harvesting chlorophyll a/b protein can be isolated from a sodium lauryl sulfate extract of chloroplasts by affinity chromatography using the immobilized light-harvesting chlorophyll a/b protein.     

Light-harvesting antenna function of phycoerythrin in prochlorococcus marinus

Prochlorococcus marinus strain CCMP 1375 is the sole prokaryote to possess phycoerythrin in addition to (divinyl-)chlorophyll a/b binding antenna complexes. Here we demonstrate, employing a spectrofluorimetric assay, that phycoerythrin serves a light-harvesting antenna function (transfers energy to chlorophylls).     

The divinyl-chlorophyll a/b-protein complexes of two strains of the oxyphototrophic marine prokaryote Prochlorococcus – characterization and response to changes in growth irradiance

Apoproteins of the antenna complexes of Prochlorococcus marinus clone SS120 (= CCMP 1375) and Prochlorococcus sp. clone MED4 (= CCMP 1378) cross-reacted with an antibody against the 30 kDa CP 5 complex of Prochlorothrix hollandica antenna. For the MED4 strain, which has a high divinyl-chlorophyll a to divinyl-chlorophyll b (DV-Chl a/b) ratio ranging from 11.4 to 15.0 (w/w), the major antenna proteins had an apparent molecular mass of 32.5 kDa. In contrast for the SS120 strain, which has a low DV-Chl a/b ratio ranging from 1.1 to 2.2, antenna apoproteins were observed in the range 34–38 kDa. For both strains, these apoproteins decreased at high growth irradiance but more markedly in the latter. Partially purified antenna fractions had a DV-Chl a/b ratio ca. 7-fold lower for SS120 than for MED4 at 30 mol photons m-2 s-1. For both strains, the 77 K fluorescence emission spectra of whole thylakoids displayed a major peak at 685 nm and a broad but very low shoulder above 700 nm. Energetic coupling of the antenna to both PS II and PSI reaction centers was demonstrated for SS120 by the strong contribution of DV-Chl b in both the 77 K excitation fluorescence spectra and the oxidized minus reduced absorption difference spectra of P700. The PS I to PS II ratio of Prochlorococcus SS120 was determined as being 0.7 ± 0.1 at low light.     

PRESENCE OF PHYCOERYTHRIN IN TWO STRAINS OF PROCHLOROCOCCUS (CYANOBACTERIA) ISOLATED FROM THE SUBTROPICAL NORTH PACIFIC OCEAN

Prochlorococcus is a ubiquitous marine oxyphotobacterium characterized by the presence of DV-chl a and b . In addition, the type strain Prochlorococcus marinus Chisholm et al. CCMP 1375 (or SS120), an isolate from the Sargasso Sea, contains low levels of an unusual phycoerythrin. Until now, it has been unclear if phycoerythrin occurs randomly within this systematic group and if the molecular characteristics of this phycoerythrin are restricted to this single strain. Here, we show that two additional Prochlorococcus strains from the Pacific Ocean also contain similar low levels of phycoerythrin. DNA sequence and phylogenetic analyses demonstrated that this phycoerythrin is very similar to the phycoerythrin of P. marinus SS120 and differs from the classic cyanobacterial phycoerythrins. In contrast, a third isolate from the Arabian Sea lacks phycoerythrin. Based on the DV-chl b:a ratio and 16S rRNA sequence data, we classify the two Pacific phycoerythrin-containing isolates as low-light-adapted strains and the Arabian Sea isolate as a high-light-adapted strain. Thus, we provide further evidence to link the physiology of an individual genotype and the presence or absence of functional phycoerythrin genes within the genus Prochlorococcus .     

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay.

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.     

Picosecond time-resolved fluorescence study of chlorophyll organisation and excitation energy distribution in chloroplasts from wild-type barley and a mutant lacking chlorophyll b.

Picosecond time-resolved fluorescence spectroscopy has been used to investigate the fluorescence emission from wild-type barley chloroplasts and from chloroplasts of the barley mutant, chlorina f-2, which lacks the light-harvesting chlorophyll a/b-protein complex. Cation-controlled regulation of the distribution of excitation energy was studied in isolated chloroplasts at the Fo and Fm levels. It was found that: (a) The fluorescence decay curves were distinctly non-exponential, even at low excitation intensities (less than 2 x 10(14) photons . cm(-2). (b) The fluorescence decay curves could, however, be described by a dual exponential decay law. The wild-type barley chloroplasts gave a short-lived fluorescence component of approximately 140 ps and a long-lived component of 600 ps (Fo) or 1300 ps (Fm) in the presence of Mg2+; in comparison, the mutant barley yielded a short-lived fluorescence component of approx. 50 ps and a long-lived component of 194 ps (Fo) and 424 ps (Fm). (c) The absence of the light-harvesting chlorophyll a/b-protein complex in the mutant results in a low fluorescence quantum yield which is unaffected by the cation composition of the medium. (d) The fluorescence yield changes seen in steady-state experiments on closing Photosystem II reaction centres (Fm/Fo) or on the addition of MgCl2 (+Mg2+/-Mg2+) were in overall agreement with those calculated from the time-resolved fluorescence measurements. The results suggest that the short-lived fluorescence component is partly attributable to the chlorophyll a antenna of Photosystem I, and, in part, to those light-harvesting-Photosystem II pigment combinations which are strongly coupled to the Photosystem I antenna chlorophyll. The long-lived fluorescence component can be ascribed to the light-harvesting-Photosystem II pigment combinations not coupled with the antenna of Photosystem I. In the case of the mutant, the two components appear to be the separate emissions from the Photosystem I and Photosystem II antenna chlorophylls.     

Thermodynamics of the beta(2) association in light-harvesting complex I of Rhodospirillum rubrum. Implication of peptide identity in dimer stability

The core light-harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n-octyl-beta-D-glucopyranoside into smaller subunit forms, exhibiting a dramatic blue-shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n-octyl-beta-D-glucopyranoside, we have previously shown that the B820 form is not only constituted by the alphabeta heterodimer alone, but that it exists in an equilibrium between the alphabeta heterodimer and beta(2) homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoretical model for alphabeta and beta(2), we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non-covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.     

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).     

Excimer fluorescence of pyrene-tropomyosin adducts

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct: and about 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35 and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the micro-environment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.     

The fluorescence decay of tryptophan residues in native and denatured proteins.

The fluorescence decay kinetics at different ranges of the emission spectrum is reported for 17 proteins. Out of eight proteins containing a single tryptophan residue per molecule, seven proteins display multiexponential decay kinetics, suggesting that variability in protein structure may exist for most proteins. Tryptophan residues whose fluorescence spectrum is red shifted may have lifetimes longer than 7 ns. Such long lifetimes have not been detected in any of the denatured proteins studied, indicating that in native proteins the tryptophans having a red-shifted spectrum are affected by the tertiary structure of the protein. The fluorescence decay kinetics of ten denatured proteins studied obey multiexponential decay functions. It is therefore concluded that the tryptophan residues in denatured proteins can be grouped in two classes. The first characterized by a relatively long lifetime of about 4 ns and the second has a short lifetime of about 1.5 ns. The emission spectrum of the group which is characterized by the longer lifetime is red shifted relative to the emission spectrum of the group characterized by the shorter lifetime. A comparison of the decay data with the quantum yield of the proteins raises the possibility that a subgroup of the tryptophan residues is fully quenched. It is noteworthy that despite this heterogeneity in the environment of tryptophan residues in each denatured protein, almost the same decay kinetics has been obtained for all the denatured proteins studied in spite of the vastly different primary structures. It is therefore concluded that each tryptophan residue interacts in a more-or-less random manner with other groups on the polypeptide chain, and that on the average the different tryptophan residues in denatured proteins have a similar type of environment.     

Lifetime of fluorescence from light-harvesting chlorophyll a/b proteins. Excitation intensity dependence.

The fluorescence from a purified, aggregate form of the light-harvesting chlorophyll a/b protein has a lifetime of 1.2 +/- 0.5 ns at low excitation intensity, but the lifetime decreases significantly when the intensity of the 20-ps, 530-nm excitation pulse is increased above about 10(16) photons/cm2. A solubilized, monomeric form of the protein, on the other hand, has a fluorescence lifetime of 3.1 +/- 0.3 ns independent of excitation intensity from 10(14)-10(18) photons/cm2/pulse. We interpret the lifetime shortening in the aggregates and the lack of shortening in monomers in terms of exciton annihilation, facilitated in the aggregate by the larger population of interacting chlorophylls.     

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm^− 1. In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Fluorescence Decaying Kinetics and Photodamage of Quinone-Reconstituted D1/D2/Cytochrome b559 Complex

Photosystem Ⅱ reaction center D1/D2/Cytochrome b559 complex loses its bound secondary electron acceptor QA and QB during isolation and purification. The artificial plastoquinone can reconstitute with the complex. The reconstitution of decyl-plastoquinone (DPQ) with D1/D2/Cytochrome b559 complex results in a decrease of the fraction of the two long lived fluorescence decay components (24 ns and 73 ns) coupled with photochemical activities to the total fluorescence yields, as well as a decrease of the total fluorescence intensity and a blue-shift of maximum emission wavelength. These results suggest that as the electron acceptor of reduced Pheo, DPQ restricts the charge recombination of P680+ Pheo-, and the two long lived fluorescence decay components (24 ns and 73 ns) come from the recombination. Although DPQ reconstitution has little effect on the susceptibility of Chi a to photodamage, β-carotene can easily be photodamaged after DPQ reconstitution. This is probably related to the physiological function of β-carotene.     

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the F?rster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.     

The Role of Acyl Lipids in Reconstitution of Lipid-Depleted Light-Harvesting Complex II from Cold-Hardened and Nonhardened Rye

The role of acyl lipids in the in vitro stabilization of the oligomeric form of light-harvesting complex II of winter rye (Secale cereale L. cv Muskateer) grown at 5 or 20°C was investigated. Purified light-harvesting complex II was enzymically delipidated to various extents by treatment with the following lipolytic enzymes: phospholipase C, phospholipase A₂, and galactolipase. Complete removal of phosphatidylcholine had no effect on the stability of the oligomeric form, whereas the removal of phosphatidylcholine plus phosphatidylglycerol caused a decrease in the ratio of oligomeric:monomeric forms from 1.86 ± 0.17 to 0.85 ± 0.17 and 3.51 ± 0.82 to 0.81 ± 0.29 for purified cold-hardened and nonhardened light-harvesting complex II, respectively, with no change in free pigment content. Incubation of delipidated cold-hardened or nonhardened light-harvesting complex with purified thylakoid phosphatidylglycerol containing trans-Δ³-hexadecenoic acid resulted in 48% reconstitution of the oligomeric form on a total chlorophyll basis with an oligomer:monomer of about 1.90. Incubation in the presence of di- 16:0 or di- 18:1 phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglyceride, or digalactosyldiacylglyceride caused no oligomerization, but rather a further destabilization of the monomeric form. These lipid-dependent structural changes were correlated with significant changes in the 77K fluorescence emission spectra for purified light-harvesting complex II. We conclude that the stabilization of the supramolecular organization of light-harvesting complex II from rye is specifically dependent upon molecular species of phosphatidylglycerol containing trans-Δ³-hexadecenoic acid.     

Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II

The fast fluorescence decay kinetics of two photosynthetic mutants of corn (Zea mays) have been compared with those of normal corn. The fluorescence of normal corn can be resolved into three exponential decay components of lifetime 900–1500 ps (slow), 300–500 ps (middle) and 50–120 ps (fast), the yields of which are affected by light intensity and Mg²⁺ levels. The Photosystem II-(PS II)-defective mutant hcf-3 has similar decay lifetimes (approx. 1200, 450 and 100 ps) but is not affected by light intensity, reflecting the absence of PS II charge recombination. However, yields do respond to Mg²⁺ in a fashion typical of normal corn, which may be correlated with the presence of normal levels of light-harvesting chlorophyll a + b complex (LHCP). The PS I mutant hcf-50 also shows three-component decay kinetics. In conjunction with the results on the LHCP-deficient mutant of barley presented in a recent paper (Karukstis, K.K. and Sauer, K. (1984) Biochim. Biophys. Acta 766, 148–155), these data suggest that the slow component of normal chloroplasts is kinetically controlled by the decay processes of the LHCP and that the energy comes from one of two sources: (a) charge recombination in the reaction centre or (b) energy transferred within or between LHCP units only. The fast component appears to originate from both PS I and PS II. The complex response of the middle component to cations and light intensity, and its presence in all of the mutants, suggests that it also may have multiple origins.     

Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence

Time-resolved fluorescence studies were carried out on the FAD bound to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the 50-500-ps time region and a longer one in the range 1.5-3.5 ns. The shorter-lifetime component has a larger contribution in the presence of substrate (p-hydroxybenzoate) or inhibitor (p-aminobenzoate). The quenching of the fluorescence is both static and dynamic in nature. The decay of fluorescence anisotropy shows that the FAD environment is both flexible and rigid. The FAD mobility can be enhanced by dilution of the enzyme, by raising the temperature, or by the binding of substrate or inhibitors. The anisotropy results are interpreted in part in terms of a monomer-dimer equilibrium, whereby the FAD in the monomer contains much more flexibility. The above-mentioned effects induce a shift of the equilibrium to the monomeric side. From a constrained parameter fitting the dissociation constant is estimated to be about 1 microM for the free enzyme and somewhat higher for the binary complexes between the enzyme and substrate or inhibitor. pH variation has only a slight effect on fluorescence or anisotropy decay parameters, while dimethylsulfoxide appears to promote dissociation into monomers by weakening hydrophobic interaction between the subunits. The results are discussed in the light of newly developed insights into the functional role of rapid structural fluctuations in enzyme catalysis.