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The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigmentâ€"protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S]2+/1+ cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g â 2 radical from P800+• following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = 3/2 ground spin state of FXâ€", and (iv) the loss of the spin-correlated P800+ FXâ€" radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of âs'502 ± 3 mV (n = 0.99), â?'496 ± 2 mV (n = 0.99), â?'517 ± 10 mV (n = 0.65), and â?'501 ± 4 mV (n = 0.84), respectively, with a consensus value of â?'504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (~15 ms) P800+ FXâ€" state is replaced by a rapidly recombining (~15 ns) P800+A0â€" state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800+ A1â€" spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S]2+/1+ clusters in the low-molecular mass ferredoxins were found to be â?'480 ± 11 mV/â€"524 ± 13 mV for PshBI, â?'453 ± 6 mV/â€"527 ± 6 mV for PshBII, and â?'452 ± 5 mV/â€"533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S]2+/1+ cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be â?'854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150â€"200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential ironâ€"sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor. [ABSTRACT FROM AUTHOR]

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection. [ABSTRACT FROM AUTHOR]

The metal sites of electron transfer proteins are tuned for function. The type 1 copper site is one of the most utilized metal sites in electron transfer reactions. This site can be tuned by the protein environment from +80 mV to +680 mV in typical type 1 sites. Accompanying this huge variation in midpoint potentials are large changes in electronic structure, resulting in proteins that are blue, green, or even red. Here, we report a family of blue copper proteins, the auracyanins, from the filamentous anoxygenic phototroph Chloroflexus aurantiacus that display die entire known spectral and redox variations known in the type 1 copper site. C aurantiacus encodes four auracyanins, labeled A-D. The midpoint potentials vary from +83 mV (auracyanin D) to +423 mV (auracyanin C). The electronic structures vary from classical blue copper UV-vis absorption spectra (auracyanin B) to highly perturbed spectra (auracyanins C and D). The spectrum of auracyanin C is temperature-dependent. The expansion and divergent nature of the auracyanins is a previously unseen phenomenon. [ABSTRACT FROM AUTHOR]

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacteriumAllochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22°C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for theTch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidumflavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins. [ABSTRACT FROM AUTHOR]

Unnatural bacteriochlorophyll (BChI) c deriv- atives possessing a hydroxy group at the terminus of a hydrocarbon chain at the 17-propionate were biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculunt tepidum. Addition of exogenous 1-octanediol, 112-dodeca- nediol, and t,16-hexadecanediol in acetone to liquid cultures resulted in accumulation of BCIII c monoesterified with the corresponding diols, The relative ratios tsf the novel BChI derivatives esterifled with 1,8-, 1,12-, and 1,16-diols to totally producing Dclii c were 8.2, 50.2, and 57.6% in the cells grown with additive a,w-diols at concentrations of 1.5, 006, and 0.06 mM, respectively, at the final concentration. The homologue composition of BChI c derivatives esterifieci with these a,es-diols was similar to that of original, coexisting BChI c esterified with farnesol (BChI CF), suggesting that esterification of a,es-diols occurred at the last step of the BChI c biosynthetic pathway by BChI synthase, BchlC, us the same manner as in BCh1 cF. Chlorosomes, which were isolated from cells grown iii the presence of exogenous a,us-diols, contained a ratio and a composition of BChl c derivatives esterified with the diols similar to those in the whole cells, indicating that these BChI c derivatives were actually present in chlorosomes Qγ absorption bands of C. tepidum cells containing the novel BCIsI c derivatives were shifted to a shorter wavelength, although their bandwidths were analogous to those of cells obtained by normal cultivation. Circular dichroism spectra of cells that had BChI c derivatives esterified with α,ω-diols exhibited S-shaped signals in the Qγregion, whose polarities were the reverse of those of cells grown in the normal medium and by supplementation with neat acetone as a control experiment. These ipectral features of C. tepidum possessing BChI derivatives estentied with α,ω-thols imply that the novel BChI c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain affect their self-assembly in chlorosomes. [ABSTRACT FROM AUTHOR]

Roseobacter denitrificans is a marine bacterium capable of using a wide variety of different metabolic schemes and in particular is an anoxygenic aerobic photosynthetic bacterium. In the work reported here we use a deletion mutant that we have constructed to investigate the structural origin of the unusual High-800 light-harvesting complex absorption in this bacterium. We suggest that the structure is essentially unaltered when compared to the usual nonameric complexes but that a change in the environment of the C13:1 carbonyl group is responsible for the change in spectrum. We tentatively relate this change to the presence of a serine residue in the a-polypeptide. Surprisingly, the low spectral overlap between the peripheral and core light-harvesting systems appears not to compromise energy collection efficiency too severely. We suggest that this may be at the expense of maintaining a low antenna size. [ABSTRACT FROM AUTHOR]

The green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum (previously known as Chlorobium tepidum), which grows at an optimal temperature of around 45 °C, biosynthesized unique disaccharide rhamnosylgalactosyldiacylglyceride (RGDG) having a methylene-bridged palmitoleyl (17:Cyc) and a palmitoyl group (16:0) as the two acyl chains in a molecule [RGDG(17:Cyc,16:0)], together with the corresponding monosaccharide monogalactosyldiacylglyceride (MGDG). Here, we report changes in the structure and composition of the glycolipids that are dependent upon the temperature and period of cultivation. With a decrease in temperature to 25 °C, the two major glycolipids were almost completely eliminated, and MGDG with a palmitoleyl (16:1) and a (16:0) group concomitantly became the major glycolipid. MGDG(16:1,16:0) corresponded to the removal of an α-rhamnosyl and a cyclopropyl methylene group from RGDG(17:Cyc,16:0) and the lack of the CH2 group in MGDG(17:Cyc,16:0). The structural conversion was almost reversible when the Cba. tepidum adapted to low and high temperatures was cultured again at 45 and 25 °C, respectively. Moreover, during this cultivation, the structure and composition of glycolipids were sequentially changed: MGDG(16:1,16:0), MGDG(17:Cyc,16:0), and RGDG(17:Cyc,16:0) predominated in the exponential, stationary and late phases of the cultivation, respectively. On the basis of these time-dependent changes, the unique disaccharide RGDG(17:Cyc,16:0) was thought to be created by the site-specific transfer of an α-rhamnosyl group to MGDG(17:Cyc,16:0) after insertion of a methylene group into the precursor MGDG(16:1,16:0). These culturing temperature- and time-dependent changes in glycolipids at the molecular level allow us to discuss their biosynthesis as well as physiological function in green photosynthetic bacteria. [ABSTRACT FROM AUTHOR]

Thermochromatium tepidum is a purple sulfur photosynthetic bacterium, and its light-harvesting 1 reaction center (LH1RC) complexes exhibit an unusual LH1 Qy absorption at 915 nm (B915) and possess enhanced thermal stability. These unique properties are closely related to an inorganic cofactor, Ca2+. Here, we report a spectroscopic variant of LH1RC complexes from Tch. tepidum cells in which Ca2+ was biosynthetically replaced with Sr2+. The photosynthetic growth of wild-type cells cannot be maintained without Ca2+ and is heavily inhibited when the Ca2+ is replaced with other metal cations. Interestingly, only Sr2+ supported photosynthetic growth instead of Ca2+ with slightly reduced rates. The resulting Sr-tepidum cells exhibited characteristic absorption spectra in the LH1 Qy region with different LH1RC:LH2 ratios depending on the growth conditions. LH1RC complexes purified from the Sr-tepidum cells exhibited a Qy maximum at 888 nm (B888) that was blue-shifted after removal of Sr2+ to 870 nm (B870). Reconstitution of Sr2+ and Ca2+ into B870 resulted in red shifts of the Qy peak to 888 and 908 nm, respectively. The thermal stability of B888 was slightly lower than that of B915 as revealed by differential scanning calorimetry analysis. Effects of other divalent metal cations on the Qy peak position and thermal stability of B888 were similar but not identical to those of B915. This study provides the first evidence of a purple bacterium in which LH1RC complexes alter spectroscopic and thermodynamic properties in vivo by utilizing exogenous metal cations and improve the ability to adapt to the environmental changes. [ABSTRACT FROM AUTHOR]

Cytochrome (cyt) bc1 is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c1 subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc1 using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc1 structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc1 subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc1 was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Qo) and quinone reduction (Qi) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Qo or Qi) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc1 preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential bL hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc1. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms have evolved diverse light-harvesting complexes to harness light of various qualities and intensities. Photosynthetic bacteria can have (bacterio)chlorophyll Qy antenna absorption bands ranging from ∼650 to ∼1100 nm. This broad range of wavelengths has allowed many organisms to thrive in unique light environments. Roseiflexus castenholzii is a niche-adapted, filamentous anoxygenic phototroph (FAP) that lacks chlorosomes, the dominant antenna found in most green bacteria, and here we describe the purification of a full complement of photosynthetic complexes: the light-harvesting (LH) antenna, reaction center (RC), and core complex (RC-LH). By high-performance liquid chromatography separation of bacteriochlorophyll and bacteriopheophytin pigments extracted from the core complex and the RC, the number of subunits that comprise the antenna was determined to be 15 ± 1. Resonance Raman spectroscopy of the carbonyl stretching region displayed modes indicating that 3C-acetyl groups of BChl a are all involved in molecular interactions probably similar to those found in LH1 complexes from purple photosynthetic bacteria. Finally, two-dimensional projections of negatively stained core complexes and the LH antenna revealed a closed, slightly elliptical LH ring with an average diameter of 130 ± 10 A surrounding a single RC that lacks an H-subunit but is associated with a tetraheme c-type cytochrome. [ABSTRACT FROM AUTHOR]

The green photosynthetic bacterium Chioroflexus auranhiacus, which belongs to the phylum of filamentous anoxygenic phototrophs. does not contain a cytochrome bc or bf type complex which is found in all other known groups of phototrophs. This suggests that a functional replacement exists to link the reaction center photochemistry to cyclic electron transfer as well as respiration. Earlier work identified a potential substitute of the cytochrome be complex, now named alternative complex Ill (ACII I), which has been purified from C. aurantiacus. identified, and characterized. ACIII functions as a menaquinol:auracyanin oxido- reductase in the photosynthetic electron transfer chain, and a related but distinct complex functions in respiratory electron flow to a terminal oxidase. In this work, we focus on elucidating the structure of photosynthetic ACIII. We found that ACIII is an integral membrane protein complex of -3OO kDa that consists of eight subunits of seven different types. Among them, there are four metalloprotein subunits, including a 113 kDa iron-sulfur cluster-containing polypeptide, a 25 kDa penta-heme c-containing subunit, and two 20 kDa monoheme c-containing subunits in the form of a homodimer. A variety of analytical techniques were employed in determining the ACIII substructure, including HPLC combined with ESI-MS, metal analysis, potentiometric titration, and intensity analysis of heme staining SDS-PAGE. A preliminary structural model ofAClil is proposed on the basis of the analytical data and chemical cross-linking in tandem with mass analysis using MALDI-TOF, as well as transmembrane and transit peptide analysis. [ABSTRACT FROM AUTHOR]

The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site- specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34C or the engineered C75C residue of wild-type PsaC (PsaCWT). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P700-FX/PsaC complex, and the P700-FX/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaCWT binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P700-FX/PsaCWT-C34 complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34C. Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaCC-term) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P700-FX/PsaCC-term-C34 complex. Because the PsaCC-term. variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180°-rotated, C2-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core. [ABSTRACT FROM AUTHOR]

The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c2 that shuttle between the reaction center and the bc1 complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c2 and bc1 complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc1 complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc1 complexes, leading to shorter pathways of cytochrome c2 between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc1 complex. [ABSTRACT FROM AUTHOR]

We investigated the effects of elevated O[sub2] pressure on the production of O[sub2] by photosynthetic organisms in several species of plants, algae, and a cyanobacteriuni. Using a noninvasive fluorornetry technique to monitor sequential turnover of the photosystem II (PSII) reaction center as a function of O[sub2] pressures, we showed that none of the reactions of water oxidation are affected by elevated O[sub2] pressures up to 50-fold greater than atmospheric conditions. Thus, the terminal step of O[sub2] release from the water oxidation complex (S[sub4] → S[sub0] + O[sub2] + nH[sup+]) is not reversible in whole cells, leaves, or isolated thylakoid membranes containing PSII, in contrast to reports using detergent-extracted PSII complexes. This implies that there is no thermodynamically accessible intermediate that can be populated by preventing or reversing the O[sub2] release step with O[sub2] at atmospheric pressure. To assess the sensitivity of PSII charge recombination to O[sub2] pressure, we quantitatively modeled the consequences of two putative perturbations to the catalytic cycle of water oxidation within the framework of the Kok model. On the basis of the breadth of oxygenic phototrophs examined in this study, we conclude that O[sub2] accumulation in cells or the atmosphere does not suppress photosynthetic productivity through the reversal of water oxidation in contemporary phototrophs and would have been unlikely to influence the evolution of oxygenic photosynthesis. [ABSTRACT FROM AUTHOR]

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp → Ala/Ala (one mutant carrying, in addition, the M249Ala → Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, , and the dynamical structure factor, S(Q,ω), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher ⟨u2 ⟩ values or the notable quasielastic broadening of S(Q,ω) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein. [ABSTRACT FROM AUTHOR]

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 µs. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This JR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741 - 13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA-QB/QAQB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA-X+/QAX spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA-QB/QAQB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs. [ABSTRACT FROM AUTHOR]

In addition to the roles of antioxidant and spacer, carotenoids (Cars) in purple photosynthetic bacteria pursue two physiological functions, i.e., light harvesting and photoprotection. To reveal the mechanisms of the photoprotective function, i.e., quenching triplet bacteriochlorophyll to prevent the sensitized generation of singlet oxygen, the triplet absorption spectra were recorded for Cars, where the number of conjugated double bonds (n) is in the region of 9-13, to determine the dependence on n of the triplet lifetime. The Cars examined include those in (a) solution; (b) the reconstituted LH1 complexes; (c) the native LH2 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, and Rps. acidophila 10050; (d) the RCs from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, and Rsp. rubrum S1; and (e) the RC-LH1 complexes from Rba. sphaeroides G1C, Rba. sphaeroides 2.4.1, Rsp. molischianum, Rps. acidophila 10050, and Rsp. rubrum S1. The results lead us to propose the following mechanisms: (i) A substantial shift of the linear dependence to shorter lifetimes on going from solution to the LH2 complex was ascribed to the twisting of the Car conjugated chain. (ii) A substantial decrease in the slope of the linear dependence on going from the reconstituted LH1 to the LH1 component of the RC-LH1 complex was ascribed to the minor-component Car forming a leak channel of triplet energy. (iii) The loss of conjugation-length dependence on going from the isolated RC to the RC component of the RC-LH1 complex was ascribed to the presence of a triplet-energy reservoir consisting of bacteriochlorophylls in the RC component. [ABSTRACT FROM AUTHOR]

The PpsR protein is a regulator of redox-dependent photosystem development in purple phototrophic bacteria. In contrast to most species, Rhodopseudomonas palustris contains two ppsR genes. We show that the inactivation of each of the R. palustris strain CGA009 ppsR genes results in an elevated level of formation of the photosystem under dark aerobic conditions. Absorption spectra of the two PpsR mutants revealed qualitative and quantitative differences in light-harvesting peak amplitude increases. A sequence difference in the helix-turn-helix DNA binding motif of PpsR2 (Arg 439 to Cys) between R. palustris strains CEA001 and CGA009 is shown to be a natural polymorphism that does not inactivate the repressor activity of the protein. To evaluate which photosynthesis genes are regulated by the two PpsR proteins, transcriptome profiles of the CGA009 and PpsR mutant strains were analyzed in microarray experiments. Transcription of most but not all photosystem genes was derepressed in the mutant strains to levels consistent with the in vivo absorption spectra, mathematical analyses of peak shapes and amplitudes, reaction center protein levels, and real-time PCR of selected mRNAs. Closely spaced PpsR binding motif repeats were identified 5′ of genes that were derepressed in the transcriptome analysis of PpsR mutants. This work shows that both the PpsR1 and PpsR2 proteins from R. palustris strain CGA009 function as oxygen-responsive transcriptional repressors. [ABSTRACT FROM AUTHOR]

The ability of chlorophylls to coordinate ligands is of fundamental structural importance for photosynthetic pigment-protein complexes, where in virtually all cases the pigment is thought to be in a pentacoordinated state. In this study, the correlation of the QX transition energy with the coordination state of the central metal in bacteriochlorophyll is applied in investigating the pigment coordination state in bacterial photosynthetic antenna LH1. To facilitate a detailed spectral analysis in the QX region, carotenoid-depleted forms of LH1 are prepared and model LH1 are constructed with non-native carotenoids having blue-shifted absorption. The deconvolution of the QX envelope in LH1 reveals that the band is the sum of two transitions, which peak near 590 and 607 nm, showing that a significant fraction (up to 25%) of hexacoordinated bacteriochlorophyll is present in the complex. The hexacoordination can be seen also in LH1 antennae from other species of purple photosynthetic bacteria. It seems correlated with the LH1 aggregation state and probably is a consequence of the structural flexibility of the assembled complex. The sixth ligand probably originates from the apoprotein and seems not to affect the chromophore core size. These findings show that in light-harvesting complexes a hexacoordinated state of bacteriochlorophyll is not uncommon. Its presence may be relevant to a correct assembly of the antenna and have functional consequences, as it results in a splitting of the pigment S2 excited state (QX), i.e., the carotenoid excitation acceptor state, what might affect intracomplex carotenoid-to-bacteriochlorophyll energy transfer. [ABSTRACT FROM AUTHOR]

Phototropic (photo) is a blue-light photoreceptor for phototropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropic has two chromophore-binding domains named LOV1 and LOV2 in its N-terminal half, each of which binds a flavin mono nucleotide (FMN) noncovalently. The C-terminal half is a Sera/Thru kinase. A transgenic study of Arabidopsis suggested that only LOV2 domain is necessary for the kinase activity, whereas X-ray crystallographic structures of LOV1 and LOV2 domains are almost identical. These facts imply that the detailed structures and/or structural changes are different between LOV1 and LOV2 domains. In this study, we compared light-induced structural changes of the LOV1 and LOV2 domains of aphototropin, Adiantum phytochrome (phy3), by means of UV- visible and Fourier transform infrared (FTIIt) spectroscopy. Photochemical properties of an adduct formation between FMN and a cysteine are essentially similar between phy3-LOV1 and phy3-LOV2. On the other hand, the S-il group of the reactive cysteine forms a hydrogen bond in phy3-LOV1, which is strengthened at low temperatures. This is possibly correlated with the fact that no adduct formation takes place for phy3-LOV1 at 77 K as revealed by the UV-visible absorption spectra. The most prominent difference was seen in the amide-I vibration that monitors the secondary structure of peptide backbone. Protein structural changes in phy3-LOV2 involve the regions of loops, a-helices, and /3-sheets, which differ significantly among various temperatures. Extended protein structural changes are probably correlated with the signal transduction activity of LOV2. In contrast, protein structural changes were very small in phy3-LOV1, and they were almost temperature independent. The photocycle of phy3-LOV1 takes 3.1 h, being more than 100 times longer than that of phy3-LOV2. These facts suggest that Adiantum phy3- LOV1 does not work for light sensing, being consistent with the previous transgenic study of Arabidopsis. It is likely that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes. [ABSTRACT FROM AUTHOR]

The role of characteristic phospholipids of native membranes, phosphatidylcholine (PC), phosphatidylglycerol (PG), and cardiolipin (CL), was studied in the energetics of the acceptor quinone side in photosynthetic reaction centers of Rhodobacter sphaeroides. The rates of the first, kAB(1), and the second, kAB(2), electron transfer and that of the charge recombination, kBP, the free energy levels of QA-(B) and QAQB- states, and the changes of charge compensating protein relaxation were determined in RCs incorporated into artificial lipid bilayer membranes. In RCs embedded in the PC vesicle, kAB(1) and kAB(2) increased (from 3100 to 4100 s-1 and from 740 to 3300 s-1, respectively) and kBP decreased (from 0.77 to 0.39 s-1) compared to those measured in detergent at pH 7. In PG, kAB(1) and kBP decreased (to values of 710 and 0.26 s-1, respectively), while kAB(2) increased to 1506 s-1 at pH 7. The free energy between the QA-QB and QAQB- states decreased in PC and PG (δG °QA-QB→QAQB-- = -76.9 and -88.5 meV, respectively) compared to that measured in detergent (-61.8 meV). The changes of the QA/QA- redox potential measured by delayed luminescence showed (1) a differential effect of lipids whether RC incorporated in micelles or vesicles, (2) an altered binding interaction between anionic lipids and RC, (3) a direct influence of PC and PG on the free energy levels of the primary and secondary quinones probably through the intraprotein hydrogen-bonding network, and (4) a larger increase of the QA/QA- free energy in PG than in PC both in detergent micelles and in single-component vesicles. On the basis of recent structural data, implications of the binding properties of phospholipids to RC and possible interactions between lipids and electron transfer components will be discussed. [ABSTRACT FROM AUTHOR]

Reconstitution experiments with a chemically synthesized core light-harvesting (LH1) β-polypeptide analogue having 3-methylhistidine instead of histidine in the position that normally donates the coordinating ligand to bacteriochiorophyll (Bchl) have provided the experimental data needed to assign to B820 one of the two possible αβ·2Bchl pairs that are observed in the crystal structure of LH2 from Phaeospiriilum (formerly Rhodospirillum) molischianum, the one with rings Ill and V of Bchl overlapping. Consistent with the assigned structure, experimental evidence is provided to show that significant stabilizing interactions for both the subunit complex (B820) and LH1 occur between the N-terminal regions of the α- and β-polypeptides. On the basis of the results with the chemically synthesized polypeptides used in this study, along with earlier results with protease-modified polypeptides, mutants, and chemically synthesized polypeptides, the importance of a stretch of 9-13 amino acids at the N-terminal end of the α- and β-polypeptides is underscored. A progressive loss of interaction with the LH1 β-polypeptide was found as the first three N-terminal amino acids of the LH1 α-polypeptide were removed. The absence of the N-terminal formylmethionine (fMet), or conversion of the sulfur in this fMet to the sulfoxide, resulted in a decrease in LH1 formation. In addition to the removal of fMet, removal of the next two amino acids also resulted in a decrease in Kassoc for B820 formation and nearly eliminated the ability to form LH1. It is suggested that the first three amino acids (fMetTrpArg) of the LH1 α-polypeptide of Rhodospirillum rubrum form a cluster that is most likely involved in close interaction with the side chain of His -18 (see Figure 1 for numbering of amino acids) of the β-polypeptide. The results provide evidence that the folding motif of the α- and β-polypeptides in the N-terminal region observed in crystal structures of LH2 is also present in LH1 and contributes significantly to stabilizing the complex. [ABSTRACT FROM AUTHOR]

Using the bacteriochlorophyll α (Bchl) cofactors as intrinsic probes to monitor changes in membrane protein structure, we investigate the response to high-pressure of the LH2 complexes from the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas acidophila 10050. By FT-Raman spectroscopy, we demonstrate that high pressure does not induce significant distortion of the protein-bound 850 nm-absorbing bacteriochlorophyll molecules, or break of the hydrogen bond they are involved in. This indicates in particular that the oligomerization of the polypeptides is not perturbed up to 0.6 GPa. The pressure-induced changes in the Bchl absorption spectra are attributed to pigmentpigment interactions. In contrast, the loss of 800 nm-absorbing bacteriochlorophyll reflects pressureinduced alterations to the tertiary structure of the protein in proximity to the membrane/cytosol interface. This suggests that the LH2 protein does have two independent structural domains. The first domain is pressure independent and comprises mostly the C-terminal domain. The second domain located on the N-terminal side exhibits sensitivity to pressure and pH reminiscent of soluble proteins. The LH2 thus constitutes a suitable model system for studying in detail the stability of membrane-embedded hydrophobic helices and helices located at or close to the solvent/membrane interface. [ABSTRACT FROM AUTHOR]

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl β-D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11 400 ± 500 and radius of gyration of 11.0 ± 1.0 Å, corresponding to a heterodimer consisting of one pair of αβ-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30 000 to 50 000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual α- and β-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The α-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the β-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between α- and β-polypeptides in reconstitution with bacteriochlorophyll a molecules. [ABSTRACT FROM AUTHOR]

Studies the characterization and isolation of the B798 light-harvesting baseplate of the chlorosome antenna complex of the thermophilic, filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus. Energy equilibrium within the Bchl alpha absorbing regions that exhibit ultrafast kinetics; Analysis of pertinent topics and relevant issues; Implications on biochemistry.

Photosystem II (PS II) contains secondary electron-transfer paths involving cytochrome b[sub 559] (Cyt b[sub 559]), chlorophyll (Chl), and β-carotene (Car) that are active under conditions when oxygen evolution is blocked such as in inhibited samples or at low temperature. Intermediates of the secondary electrontransfer pathways of PS II core complexes from Synechocystis PCC 6803 and Synechococcus sp. and spinach PS II membranes have been investigated using low temperature near-IR spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. We present evidence that two spectroscopically distinct redoxactive carotenoids are formed upon low-temperature illumination. The Car[sup +] near-IR absorption peak varies in wavelength and width as a function of illumination temperature. Also, the rate of decay during dark incubation of the Car[sup +] peak varies as a function of wavelength. Factor analysis indicates that there are two spectral forms of Car[sup +] (Car[sub A, sup +] has an absorbance maximum of 982 nm, and Car[sub B, sup +] has an absorbance maximum of 1027 nm) that decay at different rates. In Synechocystis PS II, we observe a shift of the Car[sup +] peak to shorter wavelength when oxidized tyrosine D (Y[sub D, sup •]) is present in the sample that is explained by an electrostatic interaction between Y[sub D, sup •] and a nearby β-carotene that disfavors oxidation of Car[sub B]. The sequence of electron-transfer reactions in the secondary electron-transfer pathways of PS II is discussed in terms of a hole-hopping mechanism to attain the equilibrated state of the charge separation at low temperatures. [ABSTRACT FROM AUTHOR]

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane α-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RCM. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RCM is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RCM and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RCM and RC L proteins to provide precursors for maximal production of the RC complex. [ABSTRACT FROM AUTHOR]

The absolute values of the one-electron redox potentials of the two quinones (Q[sub A] and Q[sub B]) in bacterial photosynthetic reaction centers from Rhodobacter sphaeroides were calculated by evaluating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation at pH 7.0. The redox potential for Q[sub A] was calculated to be between -173 and -160 mV, which is close to the lowest measured values that are assumed to refer to nonequilibrated protonation patterns in the redox state Q[sub A, sup -]. The redox potential of quinone QB is found to be about 160-220 mV larger for the light-exposed than for the dark-adapted structure. These values of the redox potentials are obtained if Asp-L213 is nearly protonated (probability 0.75-1.0) before and after electron transfer from Q[sub A] to Q[sub B], while Glu-L212 is partially protonated (probability 0.6) in the initial state Q[sub A]-Q[sub B, sup 0] and fully protonated in the final state Q[sub A, sup 0]Q[sub B, sup -]. Conversely, if the charge state of the quinones is varied from Q[sub A, sup -]Q[sub B, sup 0]) to Q[sub A, sup 0]Q[sub B, sup -] corresponding to the electron transfer from Q[sub A] to Q[sub B], Asp-L213 remains protonated, while Glu-L212 changes its protonation state from 0.15 H[sup +] to fully protonated. In agreement with results from FTIR spectra, there is proton uptake at Glu-L212 going along with the electron transfer, whereas Asp-L213 does not change its protonation state. However, in our simulations Asp-L213 is considered to be protonated rather than ionized as deduced from FTIR spectra. The calculated redox potential of Q[sub A] shows little dependence on the charge state of Asp-L213, which is due to a strong coupling with the protonation state of Asp-Ml7 but increases by 50 mV if Glu-L212 changes from the ionized to the protonated charge state. Both are in agreement with fluorescence measurements observing the decay of SP[sup +]Q[sub A, sup -] in a wide pH regime... [ABSTRACT FROM AUTHOR]

Studies the heterologous production of Halorhodospira halophila holo-photoactive yellow protein (PYP) through tandem expression of the postulated biosynthetic genes. Characteristics of PYP; Genes that encode the biosynthetic enzymes.

Manganese is an essential micronutrient for many organisms. Because of its unique role in the water oxidizing activity of photosystem II, manganese is required for photosynthetic growth in plants and cyanobacteria. Here we report on the mechanism of manganese uptake in the cyanobacterium Synechocystis sp. PCC 6803. Cells grown in 9 µM manganese-containing medium accumulate up to 1 × 10[sup 8] manganese atoms/cell, bound to the outer membrane (pool A). This pool could be released by EDTA treatment. Accumulation of manganese in pool A was energized by photosynthetic electron flow. Moreover, collapsing the membrane potential resulted in the immediate release of this manganese pool. The manganese in this pool is mainly Mn(II) in a six-coordinate distorted environment. A distinctly different pool of manganese, pool B (∼1.5 × 10[sup 6] atoms/cell), could not be extracted by EDTA. Transport into pool B was light-independent and could be detected only under limiting manganese concentrations (1 nM). Evidently, manganese uptake in Synechocystis 6803 cells occurs in two steps. First, manganese accumulates in the outer membrane (pool A) in a membrane potential-dependent process. Next, manganese is transported through the inner membrane into pool B. We propose that pool A serves as a store that allows the cells to overcome transient limitations in manganese in the environment. [ABSTRACT FROM AUTHOR]

Examines the energy landscape for Q[sub A[sup -] to Q [sub B] electron transfer on bacterial photosynthetic reaction centers. Importance of quinone motion; Measurement of temperature dependence of the rate on proline mutants; Difficulty to synchronize reactions on nonphotoactive proteins.

Examines the B-branch electron transfer in a quadruple reaction center mutant of the photosynthetic bacterium Rhodobacter sphaeroides. Composition of the mutant reaction center A-branch; Replacement of bacteriopheophytin with bacteriochlorophyll; Introduction of an aspartic acid residue in D mutation.

Reports on the use of photoactive yellow protein (PYP) mutant M100L to investigate transient conformational changes of the PYP photocyle. Triggering of a negative phototactic response in the phototropic bacterium Halorhodospira halophila by light-activation of the PAS domain protein PYP; Reduction of the alpha-helical content; Monitoring of tertiary structural changes by small-angle x-ray scattering measurements.

Examines the dynamics of excitation energy transfer in the light-harvesting complexes of photosynthetic bacteria. Occurrence of photosynthetic light harvesting in life processes; Structure of the bacterial peripheral light-harvesting complex; Physical origins of excitation energy transfer.

Describes the crystal structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas Acidophila strain 7050. Attribution of the observed spectroscopic differences between B800-850 LH2 and LH3 to differences in the primary structure of the apoproteins; Importance of LH3 in modulating the light-harvesting system's characteristics.

Examines the sulfur biochemistry of photosynthetic sulfur bacteria using X-ray absorption spectroscopy. Identification and quantification of the chemical forms of sulfur in a variety of bacterial cells; Estimates of the size and density of the sulfur globules in photosynthetic bacteria; Identification of the form of sulfur that most resembles the globule sulfur.

Examines the use of light by the reaction center of Rhodobacter sphaeroides to reduce and protonate a quinone molecule. Identification of the proton pathway in bacterial reaction centers; Factors attributing to the facilitation of proton transfer to the secondary quinone.

Ivestigates the interaction of metal ions with isolated photosynthetic reaction centers from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas viridis. Coordination environments of Cu[sup 2+] sites in in the RC; Role of the Cu[sup 2+] binding sites in electron transfer between the primary and secondary quinone acceptors.

Investigates strain 970, a recently isolated species of the photosynthetic purple sulfur bacteria. Use of spectroscopic techniques in the analysis; Components of purple bacteria; Description of core antenna complex.

Describes the trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria. Conformation that allows product formation; Conformational substrates along the reaction coordinate with different transition temperatures; Exothermic, electron tunneling reaction in reaction center protein.

Studies the mechanism of proton transfer retardation caused by binding of the transition metal ion to the bacterial reaction center of Rhodobacter sphaeroides. Interaction between the bound metal ion and key protonatable residues; Relationship between ligand affinity and pH; Correlation between electron and proton transfers in the inhibited reaction center.

Investigates equilibrium and kinetic parameters for the binding of inhibitors to the Q[sub B] pocket in bacterial chromatophores of Rhodobacter capsulatus and R. spheroides. Factors involved in the reduction of lipid-soluble pool of quinone molecules; Difference between stigmatellin and terbutryn in terms of binding sites; Protonation of amino acids.

Characterizes C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II of the photosynthetic bacteria Rhodobacter sphaeroides. Removal of excess lipids in empty vesicles; Purification of oxidase-containing vesicles by metal affinity column; Factors involved in the modification of chromatographic behavior of cytochrome c.

Examines proton chemical shifts in the aggregated bacteriochlorophyll c in light harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum using nuclear magnetic resonance spectroscopy. Structural components of chlorosomes; Effect of atomic self-aggregation on carbon and proton chemical shifts.

Investigates the preferential binding of oxidized cytochrome to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Drawbacks of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions; Types of kinetic behavior for cytochrome oxidation; Use of spectrophotometry.

Characterizes the Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor. Temperature dependence of the ground-state absorbance spectrum; Effect of ionic detergents on reaction centers; Rate constant of initial electron transfer in the mutant reaction centers.