Vibronic coupling explains the ultrafast carotenoid-to-bacteriochlorophyll energy transfer in natural and artificial light harvesters.

The initial energy transfer steps in photosynthesis occur on ultrafast timescales. We analyze the carotenoid to bacteriochlorophyll energy transfer in LH2 Marichromatium purpuratum as well as in an artificial light-harvesting dyad system by using transient grating and two-dimensional electronic spectroscopy with 10 fs time resolution. We find that Förster-type models reproduce the experimentally observed 60 fs transfer times, but overestimate coupling constants, which lead to a disagreement with both linear absorption and electronic 2D-spectra. We show that a vibronic model, which treats carotenoid vibrations on both electronic ground and excited states as part of the system's Hamiltonian, reproduces all measured quantities. Importantly, the vibronic model presented here can explain the fast energy transfer rates with only moderate coupling constants, which are in agreement with structure based calculations. Counterintuitively, the vibrational levels on the carotenoid electronic ground state play the central role in the excited state population transfer to bacteriochlorophyll; resonance between the donor-acceptor energy gap and the vibrational ground state energies is the physical basis of the ultrafast energy transfer rates in these systems.

Identifying residues that cause pH-dependent reduction potentials.

The pH dependence of the reduction potential E° for a metalloprotein indicates that the protonation state of at least one residue near the redox site changes and may be important for its activity. The responsible residue is usually identified by site-specific mutagenesis, which may be time-consuming. Here, the titration of E° for Chromatium vinosum high-potential iron-sulfur protein is predicted to be in good agreement with experiment using density functional theory and Poisson-Boltzmann calculations if only the sole histidine undergoes changes in protonation. The implementation of this approach into CHARMMing, a user-friendly web-based portal, allows users to identify residues in other proteins causing similar pH dependence.

Investigation of the redox interaction between Mn-bicarbonate complexes and reaction centers from Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus.

The change in the dark reduction rate of photooxidized reaction centers (RC) of type II from three anoxygenic bacteria (Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus) having different redox potentials of the P(+)/P pair and availability of RC for exogenous electron donors was investigated upon the addition of Mn(2+) and HCO(3)(-). It was found that the dark reduction of P(870)(+) from Rb. sphaeroides R-26 is considerably accelerated upon the combined addition of 0.5 mM MnCl(2) and 30-75 mM NaHCO(3) (as a result of formation of "low-potential" complexes [Mn(HCO(3))(2)]), while MnCl(2) and NaHCO(3) added separately had no such effect. The effect is not observed either in RC from Cf. aurantiacus (probably due to the low oxidation potential of the primary electron donor, P(865), which results in thermodynamic difficulties of the redox interaction between P(865)(+) and Mn(2+)) or in RC from Ch. minutissimum (apparently due to the presence of the RC-bound cytochrome preventing the direct interaction between P(870)(+) and Mn(2+)). The absence of acceleration of the dark reduction of P(870)(+) in the RC of Rb. sphaeroides R-26 when Mn(2+) and HCO(3)(-) were replaced by Mg(2+) or Ca(2+) and by formate, oxalate, or acetate, respectively, reveals the specificity of the Mn2+-bicarbonate complexes for the redox interaction with P(+). The results of this work might be considered as experimental evidence for the hypothesis of the participation of Mn(2+) complexes in the evolutionary origin of the inorganic core of the water oxidizing complex of photosystem II.

Mechanical Unfolding Pathway of the High-Potential Iron-Sulfur Protein Revealed by Single-Molecule Atomic Force Microscopy: Toward a General Unfolding Mechanism for Iron-sulfur Proteins.

High-potential iron-sulfur proteins (HiPIPs) are an important class of metalloproteins with a [4Fe-4S] cluster coordinated by four cysteine residues. Distinct from other iron-sulfur proteins, the cluster in HiPIP has a high reduction potential, making it an essential electron carrier in bacterial photosynthesis. Here, we combined single-molecule atomic force microscopy and protein engineering techniques to investigate the mechanical unfolding mechanism of HiPIP from Chromatium tepidum (cHiPIP). We found that cHiPIP unfolds in a two-step fashion with the protein sequence sequestered by the iron-sulfur center as a stable unfolding intermediate state. The rupture of the iron-sulfur center of cHiPIP proceeds in two distinct parallel pathways; one pathway involves the concurrent rupture of multiple iron-thiolate bonds, and the other one involves the sequential rupture of the iron-thiolate bonds. This mechanistic information was further confirmed by mutational studies. We found that the rupture of the iron-thiolate bonds in reduced and oxidized cHiPIP occurred in the range of 150-180 pN at a pulling speed of 400 nm/s, similar to that measured for iron-thiolate bonds in rubredoxin and ferredoxin. Our results may have important implications for understanding the general unfolding mechanism governing iron-sulfur proteins, as well as the mechanism governing the mechanical rupture of the iron-sulfur center.

Molecular cloning and sequencing of cytochrome c' from the phototrophic purple sulfur bacterium Chromatium vinosum.

The gene for cytochrome c' from Chromatium vinosum was cloned from a HindIII-SalI digest of genomic DNA. A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method. The cytochrome c' gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein. The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space. An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Biochemical and spectroscopic characterization of the reaction center-LH1 complex and the carotenoid-containing B820 subunit of Chromatium purpuratum.

Two complexes, the reaction center light-harvesting complex 1 (RC-LH1) and the B820 subunit of the LH1, have been isolated and characterized from the purple-sulfur photosynthetic bacterium Chromatium purpuratum. The RC-LH1 consists of the B870 antenna and a P-870 RC with an associated tetraheme cytochrome. This complex can be further fractionated to yield the B820 subunit of the LH1. The C. purpuratum B820 subunit is the first isolated from a purple-sulfur bacterium. It is also the first that retains its carotenoid absorption properties. CD spectra in the Qy region of bacteriochlorophyll a in both the RC-LH1 and the B820 subunit are bathochromically shifted as compared to other such complexes. Comparison of the sequence of the LH1 beta polypeptide to other LH1 beta s reveals the presence of additional aromatic amino acids in the vicinity of both of the conserved histidines in the C. purpuratum beta polypeptide. The CD spectra of these C. purpuratum pigment-protein complexes can be interpreted in terms of exciton interaction between bacteriochlorophylls in the B820 subunit of the LH1 and in the B870, with additional spectral characteristics arising from interactions of the pigments with their protein environment.

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum.

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.

Occurrence and purification of the photoactive yellow protein of Ectothiorhodospira halophila (PYP) and of immunologically related proteins of Rhodospirillum salexigens and Chromatium salexigens and intracellular localization of PYP.

The photoactive yellow protein of Ectothiohodospira halophila (PYP) was purified to homogeneity by an advanced method and applied as an affinity ligand for the isolation of an anti-PYP IgG fraction which was used for immunoscreening. The distribution of proteins immunologically related to PYP was investigated in protein fractions of 51 strains from 38 species of non-halophilic and halophilic phototrophic and chemotrophic eubacteria and archaeobacteria. Strong immunoreactive bands indicating the presence of authentic PYP on Western blots (apparent mass 17.8 kDa) was only found in the strains of E. halophila. Additionally, two soluble proteins of Chromatium salexigens and Rhodospirillum salexigens (apparent molecular masses 16.4 and 19 kDa, respectively) cross-reacted to approx. 6% and 4%. Analyses of cell fractions of E. halophila revealed that PYP is a cytoplasmic protein.

Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein.

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

Molecular modeling studies on the proposed NaCl-induced dimerization of Chromatium vinosum high-potential iron protein.

Previous work (Dunham, W.R., Hagen, W.R., Fee, J.A., Sands, R.H., Dunbar, J.B., Humblet, C. (1991) An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mössbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions, Biochimica Biophysica Acta 1079, 253-262) suggested that under specific solution conditions and slow freezing times, samples of oxidized Chromatium vinosum (Cv) high-potential, iron-sulfur protein (HiPIP) form dimeric structures that exhibit characteristic spin-spin interaction in the EPR spectrum. In that study, it was also shown that two HiPIP molecules could approach each other along their Fe1-S4 axes to a distance of approximately 13-14 A, as required by an analysis of the spin-spin physics. This is made possible because of a flattened surface on one side of the molecule within which S4 may, depending on side-chain motions, interact with solvent (Carter, C.W., Jr., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adman, E.T., Jensen, L.H. (1972) A comparison of Fe4S4 clusters in high potential iron protein and in ferredoxin, Proc. Natl. Acad. Sci. USA 69, 3527-3529). Here we describe a computer generated, hypothetical model of this proposed dimeric structure which suggests an energetically favorable interaction between two Cv HiPIP molecules and could account for the experimental observations. Two Cv HiPIP molecules brought together along their Fe1-S4 axes and maintained at a center-to-center distance of 14 A can be rotated with respect to each other so as to create complementary interactions between two glutamine residues, two phenylalanine residues, and two leucine residues, and an energetically unfavorable interaction between two arginine residues. Energy minimization calculations using the program XPLOR indicate that this arrangement may provide an overall energetically favorable interaction between the two HiPIP molecules that is strengthened by site-specific binding of Na and Cl ions.

Atomic structure of a cytochrome c' with an unusual ligand-controlled dimer dissociation at 1.8 A resolution.

The crystallographic structure of cytochrome c' from the purple phototrophic bacterium Chromatium vinosum (CVCP) has been determined at 1.8 A resolution using multiple isomorphous replacement. The molecule is a dimer, with each 131-residue chain folding as a four-helical bundle incorporating a covalently bound heme group at the core. This structure is the third of the ubiquitous cytochromes c' to be solved and is similar to the known structures of cytochrome c' from R. molischianum (RMCP) and R. rubrum (RRCP). CVCP is unique in exhibiting ligand-controlled dimer dissociation while RMCP and RRCP do not. The Tyr16 side-chain, which replaced Met16 in RMCP and Leu14 in RRCP, is parallel to the heme plane and located directly above the sixth ligand site of the heme Fe. Any ligand binding to this site, such as CO or CN-, must move the Tyr16 side-chain, which would be expected to cause other conformational changes of helix A, which contributes to the dimer interface, and consequently disrupting the dimer. Thus, the crystallographic structure of CVCP suggests a mechanism for dimer dissociation upon ligand binding. The dimer interface specificity is due to a lock and key shape complementarity of hydrophobic residues and not to any charge complementarity or cross-interface hydrogen bonds as is common in other protein-protein interfaces. The co-ordinates have been deposited in the Brookhaven Data Bank (entry P1BBH).

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.

Covalent structure of the flavoprotein subunit of the flavocytochrome c: sulfide dehydrogenase from the purple phototrophic bacterium Chromatium vinosum.

The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c-sulfide dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three-dimensional structure determination (Chen Z et al., 1994, Science 266:430-432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 +/- 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three-dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three-dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Trp 391, Glu 167, and the positive end of a helix dipole.

Chemical crosslinking studies of the isolated light-harvesting B800-850 complex of Chromatium minutissimum.

The spatial relationship of polypeptides comprising the light-harvesting B800-850 complex of Chromatium minutissimum has been studied by means of chemical crosslinking of the isolated complex with cleavable, 1.2 nm-long dithiobis(succinimidyl propionate). The samples were analyzed by different types of electrophoresis and spectrophotometrically. No difference was shown between crosslinking of the B800-850 complex either solubilized or incorporated into proteoliposomes. It was found that two main polypeptides form only one type of heterodimer. The crosslinked complex was more thermostable. This crosslinkage restricted the conformational transitions causing the shift of the long wavelength band in the near infrared region. A structure of the complex is discussed.

Preliminary X-ray crystallographic studies of photosynthetic reaction center from a thermophilic sulfur bacterium, Chromatium tepidum.

A membrane protein complex, photosynthetic reaction center purified from the thermophilic purple sulfur bacterium, Chromatium tepidum has been crystallized from a phosphate buffer containing a detergent, n-octyl-beta-D-glucopyranoside and a precipitant, polyethylene glycol 4000. The crystals diffracted X-rays beyond 3A resolution with synchrotron radiation and are suitable for high-resolution X-ray crystallographic studies. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions of a = 136A, b = 197A, and c = 82A. Assuming that they contain one reaction center complex in the asymmetric unit, VM was calculated to be 4.3 A3/Da, which agrees with the values obtained in the membrane protein complexes.

Picosecond dynamics of excitations in light-harvesting complex B800-850 from Chromatium minutissimum studied using fluorescence spectrochronography.

The picosecond dynamics of excitations in the isolated B800-850 light-harvesting complex of the purple sulfur bacterium Chromatium minutissimum has been studied using picosecond fluorescence spectrochronography. A short-lived component of about 20 ps lifetime has been found at 77K at the short wavelength part of the B850 fluorescence spectrum similar to that previously described for the core antenna bacteriochlorophyll band B880 of Rhodospirillum rubrum. Evidence has been presented indicating that this component is likely to reflect excitation energy relaxation step(s) involving both photoexcited bacteriochlorophyll and the protein environment. A new kinetic scheme of excitation transfer from the peripheral antenna to the photoreceptor units in purple bacteria is suggested which takes into account these findings.

Excitonic interactions in the light-harvesting antenna of photosynthetic purple bacteria and their influence on picosecond absorbance difference spectra.

A new model of the light-harvesting antenna (core complex) of purple photosynthetic bacteria is proposed based on excitonic interactions in circular aggregates of bacteriochlorophyll molecules. The calculated absorbance difference spectra of circular aggregates demonstrate all special features observed in the experimental spectra of purple bacteria. In particular, the absorption changes with high amplitude of bleaching at the long-wavelength side of the absorption band at different excitation energy are predicted.

N-terminal methylation of the core light-harvesting complex in purple photosynthetic bacteria.

Several core light-harvesting complexes from both sulfur and non-sulfur purple photosynthetic bacteria have been identified to be methylated at the N-terminal alpha-amino group of beta-polypeptides by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance. Monomethylation has been confirmed for the N-terminal alanine residues of the beta-polypeptides from Rhodospirillum rubrum, Thermochromatium tepidum and Chromatium vinosum, but not for the beta-polypeptide from Rhodobacter sphaeroides. The modification appears to be related with the amino acid sequence and charge distribution in the N-terminal end. Some common features and possible functions are discussed.

The cytochrome subunit structure in the photosynthetic reaction center of Chromatium minutissimum.

Gel-electrophoretic assay revealed that the photosynthetic reaction center (RC) of Chromatium minutissimum, in contrast to the well-known RC Rhodopseudomonas viridis, consists of five rather than four subunits with molecular masses of 37, 34, 25, 19, and 17 kDa. The 37- and 19-kDa subunits are stained with tetramethylbenzidine for the cytochrome c hemes. Absorption spectra show that the concentration of reduced cytochromes in the C. minutissimum RC poised at redox potential of -150 mV (fully reduced pool of hemes) is about three times more than in the C. minutissimum RC poised at redox potential of +260 mV (only high-potential hemes are reduced). The results of redox titration of absorption changes at the cytochrome c alpha-band are most appropriately approximated by a six-component theoretical curve with the midpoint potentials of Em1 = 390 mV, Em2 = 320 mV, Em3 = 210 mV, Em4 = 100 mV, Em5 = 20 mV, and Em6 = -50 mV. Possible functions of the cytochromes with the midpoint potentials 210 and 100 mV, which have not been found in purple bacteria before, are discussed.