Biomedical subjects

H Scheer

Publications and source records attributed to H Scheer.

At least 19 recordsLinked to original sources

PubMed2002

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically 'dark') S(1) state of carotenoids.

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300-1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2A(g)(-) state of rhodopin [corrected] is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid-depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

Bacteriochlorophylls↗

PubMed2002

Assembly of light-harvesting bacteriochlorophyll in a model transmembrane helix in its natural environment.

The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins.

Amino Acid Sequence↗

PubMed2002

Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities.

Phycoerythrocyanin (PEC) is part of the light harvesting system of cyanobacteria. The PEC monomer contains one phycoviolobilin chromophore, which transfers excitation energy onto two phycocyanobilin chromophores. Many spectroscopical methods have been used in the past to study the bulk properties of PEC. These methods average over many molecules. Therefore, differences in the behavior of individual molecules remain hidden. The energy transfer within photosynthetic complexes is however sensitive to changes in the spectroscopic properties of the participating subunits. Knowledge about heterogeneities is therefore important for the description of the energy transfer in photosynthetic systems. Here, the recording of the fluorescence emission of single PEC molecules is used as a tool to obtain such information. Spectrally resolved detection as well as double resonance excitation of single PEC molecules is used to investigate their bleaching behavior. The trans isomer of the phycoviolobilin chromophore is identified as a short-lived dark state of monomeric PEC. Polarization sensitive single molecule detection is used for the direct observation of the energy transfer in individual PEC molecules. The experiments reveal that more than one-half of the PEC molecules exhibit an energy transfer behavior significantly different from the bulk. These heterogeneities persist on a time scale of several seconds. Model calculations lead to the conclusion that they are caused by minor shifts in the spectra of the chromophores.

Biophysical Phenomena↗

PubMed2001

FTIR studies of phytochrome photoreactions reveal the C=O bands of the chromophore: consequences for its protonation states, conformation, and protein interaction.

The molecular changes of phytochrome during red --> far-red and reverse photoreactions have been monitored by static infrared difference spectroscopy using the recombinant 65 kDa N-terminal fragment assembled with a chromophore chemically modified at ring D or with a chromophore isotopically labeled with (18)O at the carbonyl group of ring A. This allows the identification of the C=O stretching vibrations of rings D and A. We exclude the formation of an iminoether in Pfr. The positions of both these modes show that the chromophore always remains protonated. The upshift of the C=O stretch of ring D in the first photoproducts is explained by a twisted methine bridge connecting rings C and D. The changes in the vibrational pattern during the red --> far-red conversion show that the backreaction is not just the reversal of the forward reaction. The infrared difference spectra of the fragment deviate very little from those of the full-length protein. The differences which are related to the lack of the C-terminal half of the protein constituting the signaling domain are possibly important for the understanding of the signaling mechanism.

Avena↗

PubMed2001

Chromophore attachment to biliproteins: specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin.

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.

Amino Acid Sequence↗

PubMed2001

Excitation trap approach to analyze size and pigment-pigment coupling: reconstitution of LH1 antenna of Rhodobacter sphaeroides with Ni-substituted bacteriochlorophyll.

Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties. The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-beta-glucopyranoside (OG). Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll. By adding increasing amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap. In contrast to the nearly unchanged absorption, the presence of [Ni]-BChl in LH1 markedly affects the emission properties. Incorporation of only 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, respectively. The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 +/- 1 BChl molecules.

Bacterial Proteins↗

PubMed2001

Design, synthesis and properties of synthetic chlorophyll proteins.

A chemoselective method is described for coupling chlorophyll derivatives with an aldehyde group to synthetic peptides or proteins modified with an aminoxyacetyl group at the epsilon-amino group of a lysine residue. Three template-assembled antiparallel four-helix bundles were synthesized for the ligation of one or two chlorophylls. This was achieved by coupling unprotected peptides to cysteine residues of a cyclic decapeptide by thioether formation. The amphiphilic helices were designed to form a hydrophobic pocket for the chlorophyll derivatives. Chlorophyll derivatives Zn-methyl-pheophorbide b and Zn-methyl-pyropheophorbide d were used. The aldehyde group of these chlorophyll derivatives was ligated to the modified lysine group to form an oxime bond. The peptide-chlorophyll conjugates were characterized by electrospray mass spectrometry, analytical HPLC, and UV/visible spectroscopy. Two four-helix bundle chlorophyll conjugates were further characterized by size-exclusion chromatography, circular dichroism, and resonance Raman spectroscopy.

Amino Acid Sequence↗

PubMed2001

Photodynamics of the bacteriochlorophyll-carotenoid system. 1. Bacteriochlorophyll-photosensitized oxygenation of beta-carotene in acetone.

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 beta-carotene (Car) in aerobic acetone is rapidly oxygenated under strong illumination with red light (lambda exc > or = 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 C40-skeleton remains intact.

Acetone↗

PubMed2000

Relevance of the chlorophyll phytyl chain on lamellar phase formation and organisation.

A series of modified chlorophylls (chlorophyll a, pyrochlorophyll a, Zn-pheophytin a and Zn-pheophorbide a) have been inserted into lamellar phases of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT). The role played by the different functional groups in affecting the bilayer formation and organisation has been investigated by means of the NMR quadrupolar splitting technique. Evidence is reported for the first time on the capacity of the phytyl chain of the chlorophylls to anchor the tetrapyrroles into the bilayer, favouring at the same time the regular formation of the lamellae.

Chlorophyll↗

PubMed2000

Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon.

The structure of phycoviolobilin, the photoactive chromophore of alpha-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a Delta3, 3(1)-double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-alpha-phycoerythrocyanin (PecA). In the absence of the components of alpha-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (lambda(max)=641 nm) and low photoactivity (<10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (lambda(max)=565 nm) and high photoreversible photochromism (100% type I) has been identified as integral alpha-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.

Bacterial Proteins↗

PubMed2000

Exchanging cofactors in the core antennae from purple bacteria: structure and properties of Zn-bacteriopheophytin-containing LH1.

The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactors present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This technique makes use of the well-characterized, reversible dissociation of LH1 induced by the detergent beta-octylglucoside. Incubation of partially dissociated LH1 with exogeneous pigments induces an equilibrium between the protein-bound BChl and the exogeneous pigment. This results in the binding of chemically modified pigments to LH1, in amounts which depend on the pigment composition and concentration of the exchange buffer. This method can yield information on the relative affinities of the LH1 protein-binding sites for the different pigments BChl and Zn-Bpheo and can also be used to obtain fully reassociated LH1 proteins, with a variable content of modified pigment, which may be precisely monitored. Absorption and FT-Raman spectroscopy indicate that this exchange procedure leads to LH1 proteins containing modified pigments, but retaining a binding site structure identical to that of native LH1. Furthermore, examination of the binding curves suggests that there are two distinguishable binding sites, probably corresponding to the two polypeptides, with very different properties. One of these two binding sites shows a marked preference for Zn-Bpheo over BChl, while the other binding site appears to prefer BChl.

Bacterial Proteins↗

PubMed2000

B800-->B850 energy transfer mechanism in bacterial LH2 complexes investigated by B800 pigment exchange.

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a-B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q(y) absorption maximum can be shifted incrementally from 800 to 670 nm. As the spectral overlap between the B800 and B850 Q(y) bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9 ps (bacteriochlorophyll a in the B800 sites, Q(y) absorption maximum at 800 nm) to 8.3 ps (chlorophyll a in the B800 sites, Q(y) absorption maximum at 670 nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from Förster spectral overlap calculations, although the experimentally determined rates are approximately 5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.

Bacterial Proteins↗

PubMed2000

Photophysical consequences of coupling bacteriochlorophyll a with serine and its resulting solubility in water.

We investigated the dependence on solvents of optical absorption and emission of the bacteriochlorophyll a-serine (BChl-ser), a water soluble bacteriochlorophyll (BChl) derivative. Comparison between the experimental data and those collected for BChl in nonaqueous solvents shows that only a minor interaction takes place between serine and the macrocycle's pi-electron system. Nevertheless, the coupling with serine results in a small enhancement of the nonradiative relaxation rate from the first excited singlet state S1. In buffered aqueous solution (pH = 7.4), the Stokes shift of the BChl-ser fluorescence and its nonradiative relaxation rate are enhanced compared with those in nonaqueous solutions (Scherz, A., S. Katz, Y. Vakrat, V. Brumfeld, E. Gabelmann, D. Leupold, J. R. Norris, H. Scheer and Y. Salomon (1998) Photosynthesis: Mechanisms and Effects, Vol. V (Edited by G. Garab), pp. 4207-4212. Kluwer Academic, Dordrecht.), probably as a result of a hydrogen bonding between the BChl macrocycle and the water molecules. In aprotic solvents, without hydrogen bonds, the permanent dipole moment of the first excited singlet state in both BChl and BChl-ser is increased compared with the ground state by at least 2.5 Debye.

Bacteriochlorophylls↗

PubMed1999

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

Bacterial Proteins↗

PubMed1999

Selective release, removal, and reconstitution of bacteriochlorophyll a molecules into the B800 sites of LH2 complexes from Rhodopseudomonas acidophila 10050.

A method is described which allows the selective release and removal of the Bchla-B800 molecules from the LH2 complex of Rhodopseudomonas acidophila 10050. This procedure also allows reconstitution of approximately 80% of the empty binding sites with native Bchla. As shown by circular dichroism spectroscopy, the overall structures of the B850-only and reconstituted complexes are not affected by the pigment-exchange procedure. The pigments reconstituted into the B800 sites can also efficiently transfer excitation energy to the Bchla-B850 molecules.

Bacterial Proteins↗

PubMed1999

Bacteriochlorin-protein interactions in native B800-B850, B800 deficient and B800-Bchla(p)-reconstituted complexes from Rhodopseudomonas acidophila, strain 10050.

Recently, a method which allows the selective release and removal of the 800 nm absorbing bacteriochlorophyll a (B800) molecules from the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been described [Fraser, N.J. (1999) Ph.D. Thesis, University of Glasgow, UK]. This procedure also allows the reconstitution of empty binding sites with the native pigment Bchla(p), esterified with phytol. We have investigated the bacteriochlorophylla-protein interactions in native, B800 deficient (or B850) and in B8110-bacteriochlorophylla(p)-reconstituted LH2 complexes by resonance Raman spectroscopy. We present the first direct structural evidence which shows that the reconstituted pigments are correctly bound within their binding pockets.

Bacterial Proteins↗

PubMed1998

Biosynthesis of the 3-acetyl and 13(1)-oxo groups of bacteriochlorophyll a in the facultative aerobic bacterium, Rhodovulum sulfidophilum--the presence of both oxygenase and hydratase pathways for isocyclic ring formation.

Using (18)O-labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BCh1 a both aerobically in the dark and anaerobically in the light. R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate (18)O label from H2(18)O into both the 13(1)-oxo and 3-acetyl oxygens; in addition, the four carboxyl oxygens at C13(3) and C17(3) were labelled by H2(18)O. Under aerobic conditions in the dark, the labelling of the 13(1)-oxo group by H2(18)O was reduced indicating that (16)O was being incorporated into this group from air. R. sulfidophilum, grown in the dark under an atmosphere initially containing 50% (18)O2 in Ar, possessed an oxygenase which incorporated (18)O label from (18)O2 specifically into the 13(1)-oxo group; under these conditions the acetyl and carboxyl groups remained unlabelled. Thus, both an oxygenase and hydratase operate in R. sulfidophilum to form the 13(1)-oxo group of ring E of BCh1 a; the 3-acetyl group oxygen, however, arises only from water via a hydratase.

Bacteriochlorophylls↗

PubMed1998

Modification of pigment composition in the isolated reaction center of photosystem II.

The pigment content of isolated reaction centers of photosystem II was modified using an exchange protocol similar to that used for purple bacterial reaction centers. With this method, which is based on incubation of reaction centers at elevated temperature with an excess of chemically modified pigments, it was possible to incorporate [3-acetyl]-chlorophyll a and [Zn]-chlorophyll a into photosystem II reaction centers. Pigment exchange has been verified by absorption, circular dichroism and fluorescence spectroscopy, and quantitated by HPLC analysis of pigment extracts.

Chromatography, High Pressure Liquid↗