Modulation of fluorescence in Heliobacterium modesticaldum cells

In what appears to be a common theme for all phototrophs, heliobacteria exhibit complex modulations of fluorescence yield when illuminated with actinic light and probed on a time scale of μs to minutes. The fluorescence yield from cells of Heliobacterium modesticaldum remained nearly constant for the first 10–100 ms of illumination and then rose to a maximum level with one or two inflections over the course of many seconds. Fluorescence then declined to a steady-state value within about one minute. In this analysis, the origins of the fluorescence induction in whole cells of heliobacteria are investigated by treating cells with a combination of electron accepters, donors, and inhibitors of the photosynthetic electron transport, as well as varying the temperature. We conclude that fluorescence modulation in H. modesticaldum results from acceptor-side limitation in the reaction center (RC), possibly due to charge recombination between P₈₀₀ ⁺ and A₀ ^?.     

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center

Heliobacteria contain a very simple photosynthetic apparatus, consisting of a homodimeric type I reaction center (RC) without a peripheral antenna system and using the unique pigment bacteriochlorophyll (BChl) g. They are thought to use a light-driven cyclic electron transport pathway to pump protons, and thereby phosphorylate ADP, although some of the details of this cycle are yet to be worked out. We previously reported that the fluorescence emission from the heliobacterial RC in vivo was increased by exposure to actinic light, although this variable fluorescence phenomenon exhibited very different characteristics to that in oxygenic phototrophs (Collins et al. 2010). Here, we describe the underlying mechanism behind the variable fluorescence in heliobacterial cells. We find that the ability to stably photobleach P₈₀₀, the primary donor of the RC, using brief flashes is inversely correlated to the variable fluorescence. Using pump-probe spectroscopy in the nanosecond timescale, we found that illumination of cells with bright light for a few seconds put them in a state in which a significant fraction of the RCs underwent charge recombination from P₈₀₀ ⁺A₀ ^? with a time constant of ~20 ns. The fraction of RCs in the rapidly back-reacting state correlated very well with the variable fluorescence, indicating that nearly all of the increase in fluorescence could be explained by charge recombination of P₈₀₀ ⁺A₀ ^?, some of which regenerated the singlet excited state. This hypothesis was tested directly by time-resolved fluorescence studies in the ps and ns timescales. The major decay component in whole cells had a 20-ps decay time, representing trapping by the RC. Treatment of cells with dithionite resulted in the appearance of a ~18-ns decay component, which accounted for ~0.6 % of the decay, but was almost undetectable in the untreated cells. We conclude that strong illumination of heliobacterial cells can result in saturation of the electron acceptor pool, leading to reduction of the acceptor side of the RC and the creation of a back-reacting RC state that gives rise to delayed fluorescence.     

Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum

Cytochrome c₅₅₃ of Heliobacterium modesticaldum is the donor to P₈₀₀ ⁺, the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c ₅₅₃ was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c-type cytochrome, including an asymmetric α-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c ₅₅₃ and the HbRC was also studied. Re-reduction of photooxidized P₈₀₀ ⁺ was accelerated by addition of reduced cytochrome c ₅₅₃. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 10⁴ M^?1 s^?1 at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol^?1, similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.     

The bound iron–sulfur clusters of Type-I homodimeric reaction centers

The hallmark of a Type-I photosynthetic reaction center (RC) is the presence of three [4Fe–4S]^2+/1+ clusters, named F_X, F_A, and F_B that act as terminal electron acceptors. Their function is to increase the distance, and hence the lifetime, of the initial charge-separated state so that diffusion-mediated processes, such as the reduction of ferredoxin, can occur. Type-I homodimeric RCs, such as those found in heliobacteria, green-sulfur bacteria, and Candidatus Chloracidobacterium thermophilum, are less well understood than Photosystem I, the prototypical Type-I heterodimeric RC found in cyanobacteria and plants. Here, we review recent progress that has been made in elucidating the spectroscopic and biochemical properties of the bound Fe/S clusters and their cognate proteins in homodimeric Type-I RCs. In Heliobacterium modesticaldum, the identification and characterization of two loosely bound polypeptides, PshBI and PshBII that harbor the F_A and F_B clusters threatens to break the long-accepted assumption that Type-I RCs harbor one tightly bound F_A/F_B-containing protein. Additionally, the detection of the F_X cluster in S = 1/2 and S = 3/2 ground spin states has resolved the long-standing issue of its missing EPR spectrum. In Chlorobaculum tepidum, the focus is on the biochemical properties of the unusual extrinsic Fe/S protein, PscB, which is readily dissociable from the RC core. The C-terminal domain of PscB is constructed as a bacterial ferredoxin, harboring the F_A and F_B clusters, but the N-terminal domain contains a number of PxxP motifs and is rich in Lys, Pro, and Ala residues, features characteristic of proteins that interact with SH3 domains. Little is known about Candidatus Chloracidobacterium thermophilum except that the photosynthetic RC is predicted to be a Type-I homodimer with an F_X-binding site. These findings are placed in a context that promises to unify the acceptor side of homodimeric Type-I RCs in prokaryotic phototrophs.     

Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis>

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c ₅₅₃ and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 8¹-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P₈₀₀ ⁺F_X⁻ state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s⁻¹ under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.     

The F<Subscript>X</Subscript> iron–sulfur cluster serves as the terminal bound electron acceptor in heliobacterial reaction centers

Phototrophs of the family Heliobacteriaceae contain the simplest known Type I reaction center (RC), consisting of a homodimeric (PshA)₂ core devoid of bound cytochromes and antenna proteins. Unlike plant and cyanobacterial Photosystem I in which the F_A/F_B protein, PsaC, is tightly bound to P₇₀₀–F_X cores, the RCs of Heliobacterium modesticaldum contain two F_A/F_B proteins, PshBI and PshBII, which are loosely bound to P₈₀₀–F_X cores. These two 2[4Fe–4S] ferredoxins have been proposed to function as mobile redox proteins, reducing downstream metabolic partners much in the same manner as does [2Fe–2S] ferredoxin or flavodoxin (Fld) in PS I. Using P₈₀₀–F_X cores devoid of PshBI and PshBII, we show that iron–sulfur cluster F_X directly reduces Fld without the involvement of F_A or F_B (Fld is used as a proxy for soluble redox proteins even though a gene encoding Fld is not identified in the H. modesticaldum genome). The reduction of Fld is suppressed by the addition of PshBI or PshBII, an effect explained by competition for the electron on F_X. In contrast, P₇₀₀–F_X cores require the presence of the PsaC, and hence, the F_A/F_B clusters for Fld (or ferredoxin) reduction. Thus, in H. modesticaldum, the interpolypeptide F_X cluster serves as the terminal bound electron acceptor. This finding implies that the homodimeric (PshA)₂ cores should be capable of donating electrons to a wide variety of yet-to-be characterized soluble redox partners.     

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g_F, where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b_P, where P is phytyl, rather than the native BChl a_P, we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g_P. Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG^Rs gene from Rba. sphaeroides was replaced with bchG^Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g_F. The construction of a strain producing BChl g_F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g_F.     

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2_A/ec3_A or ec2_B/ec3_B) and a phylloquinone (PhQ_A or PhQ_B). The axial ligand to the central Mg^2 + of the ec2_A and ec2_B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to F_X electron transfer show that in both species, the PsaA-N604L mutation (near ec2_B) results in a ~ 50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2_A) results in a ~ 70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P₇₀₀⁺ PhQ^? is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.     

Effects of 28-homobrassinoloid on key physiological attributes of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> seedlings under cadmium stress: Photosynthesis and nitrogen metabolism

Heavy metal accumulation due to environmental pollution, especially in agricultural ecosystem can cause serious deterioration of crop yield and quality. In present study we assessed the effect of exogenous 28-homobrassinoloid (HBL; 10^?8 M) on growth, photosynthesis, indices of chlorophyll a fluorescence and nitrogen metabolism in Solanum lycopersicum seedlings grown under two doses (Cd₁: 3 mg kg^?1 sand and Cd₂: 9 mg kg^?1 sand) of cadmium. Accumulation of Cd in root tissues was considerably higher than shoot hence, Cd declined the growth, pigment contents, and photosynthetic O₂ yield in its concentration dependent manner. Chlorophyll a fluorescence due to Cd stress was negatively affected as shown by decreased Q_A ^? reoxidation kinetics: φP₀, ψ₀, φE₀ and PI__ABS and increased energy flux parameters: ABS/RC, TR₀/RC, ET₀/RC and DI₀/RC. HBL application under Cd stress improved the photochemistry of photosystem II (PS II) by affecting these parameters positively. Treatment of Cd in test seedlings resulted into significant decrease in nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activities, and induced enhancing effect on ammonium content and glutamate dehydrogenase activity. Exogenous HBL treatment alleviated the negative effect of Cd on growth, photosynthesis, contents of protein, carbohydrate and inorganic nitrogen and nitrogen assimilating enzymes. The data indicate that exogenous HBL protects the test seedlings during the early growth phase against Cd phytotoxicity by regulating Cd accumulation in tissues and two key metabolic processes; photosynthesis and nitrogen metabolism.     

Kinetin Regulates UV-B-Induced Damage to Growth,Photosystem II Photochemistry,and Nitrogen Metabolism in Tomato Seedlings

Cytokinins are a class of plant growth regulators that regulate several developmental processes in plants, and recently their role in counteracting the deleterious effects of abiotic stresses has been noted. The impacts of kinetin (10 µM, KN; an artificial cytokinin) on growth, photosystem II photochemistry, and nitrogen metabolism in tomato seedlings exposed to two levels (UV-B₁, ambient+?1.2 kJ m^?2 day^?1, and UV-B₂, ambient+?2.4 kJ m^?2 day^?1) of enhanced UV-B radiation were analyzed under open field condition. The growth, pigment contents, carbonic anhydrase activity, photosynthetic O₂ yield, and values of chlorophyll a fluorescence parameters: F _v/F ₀, F _v/F _m or φP₀, ψ ₀, φE ₀, and PI_ABS declined, whereas the values of energy flux parameters (ABS/RC, TR₀/RC, ET₀/RC, and DI₀/RC) of PS II, efficiency of water splitting complex (F ₀/F _v), and respiratory rate of O₂ uptake increased under UV-B stress. Likewise, UV-B exposure at both doses significantly inhibited the activity of enzymes involved in nitrogen metabolism: nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase. In contrast, an enhancing effect on glutamate dehydrogenase activity was observed under UV-B stress. Exogenous KN resulted in a significant attenuation in UV-B-induced negative effects on growth, pigments, photosynthesis, and nitrogen metabolism. The study concludes that exogenous KN improved the growth performance of tomato seedlings by attenuating the damaging effects of UV-B radiation on photochemistry of PS II and nitrogen metabolism, and the alleviating effect against the low dose (UV-B₁) of UV-B was more pronounced.     

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S₂ (1¹B _u ⁺ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S₂ (1¹B _u ⁺ ) state to BChl a via the Q_x state of the latter, accounting for 60% of the total transfer. The elevated S₂ (1¹B _u ⁺ ) → Q_x transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S₂ (1¹B _u ⁺ ) → S₀ (1¹A _g ^? ) carotenoid emission and Q_x absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.     

Completion of biosynthetic pathways for bacteriochlorophyll g in Heliobacterium modesticaldum: The C8-ethylidene group formation

Heliobacteria have the simplest photosynthetic apparatus, i.e., a type-I reaction center lacking a peripheral light-harvesting complex. Bacteriochlorophyll (BChl) g molecules are bound to the reaction center complex and work both as special-pair and antenna pigments. The C8-ethylidene group formation for BChl g is the last missing link in biosynthetic pathways for bacterial special-pair pigments, which include BChls a and b as well. Here, we report that chlorophyllide a oxidoreductase (COR) of Heliobacterium modesticaldum catalyzes the C8-ethylidene formation from 8-vinyl-chlorophyllide a, producing bacteriochlorophyllide g, the direct precursor for BChl g without the farnesyl tail. The finding led to plausible biosynthetic pathways for 8¹-hydroxy-chlorophyll a, a primary electron acceptor from the special pair in heliobacterial reaction centers. Proposed catalytic mechanisms on hydrogenation reaction of the ethylidene synthase-type CORs are also discussed.     

Identification of the Intermediate Charge-Separated State PβL in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B_L and B_M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm⁻¹ and 1770 cm⁻¹, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P^∗ decayed on a timescale of ∼6.3 ps to P⁺B_L⁻. The decay of P⁺B_L⁻ occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β_L BChl resulted in formation of the P⁺β_L⁻ state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P⁺B_L⁻ and P⁺β_L⁻ showed clear differences related to the different environments of the two BChls in the mutant RC.     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (∼ 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A₁ instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of PA and BB bacteriochlorophylls

To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P_A or BChl B_B. Contrary to expectations, the double mutation I(L177)H + H(L173)L does not bring about a heterodimer RC but causes a 46 nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for P_A in the RC I(L177)H + H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9 Å shows changes at the interface region between the BChl P_A and the monomeric BChl B_B. Spectral and pigment analysis provided evidence for β-coordination of the BChl B_B in the double mutant RC I(L177)H + H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl B_B. Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P_A and B_B are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.