The photosystem of Rhodobacter sphaeroides assembles with zinc bacteriochlorophyll in a bchD (magnesium chelatase) mutant

A Rhodobacter sphaeroides bchD (magnesium chelatase) mutant was studied to determine the properties of its photosystem in the absence of bacteriochlorophyll (BChl). Western blots of reaction center H, M, and L (RC H/M/L) proteins from mutant membranes showed levels of 12% RC H, 32% RC L, and 46% RC M relative to those of the wild type. Tricine-SDS-PAGE revealed 52% light-harvesting complex alpha chain and 14% beta chain proteins compared to those of the wild type. Pigment analysis of bchD cells showed the absence of BChl and bacteriopheophytin (BPhe), but zinc bacteriochlorophyll (Zn-BChl) was discovered. Zn-BChl binds to light-harvesting 1 (LH1) and 2 (LH2) complexes in place of BChl in bchD membranes, with a LH2:LH1 ratio resembling that of wild-type cells under BChl-limiting conditions. Furthermore, the RC from the bchD mutant contained Zn-BChl in the special pair and accessory BChl binding sites, as well as carotenoid and quinone, but BPhe was absent. Comparison of the bchD mutant RC absorption spectrum to that of Acidiphilium rubrum, which contains Zn-BChl in the RC, suggests the RC protein environment at L168 contributes to A. rubrum special pair absorption characteristics rather than solely Zn-BChl. We speculate that Zn-BChl is synthesized via the normal BChl biosynthetic pathway, but with ferrochelatase supplying zinc protoporphyrin IX for enzymatic steps following the nonfunctional magnesium chelatase. The absence of BPhe in bchD cells is likely related to Zn2+ stability in the chlorin macrocycle and consequently high resistance of Zn-BChl to pheophytinization (dechelation). Possible agents prevented from dechelating Zn-BChl include the RC itself, a hypothetical dechelatase enzyme, and spontaneous processes.     

A picosecond circular dichroism study of photosynthetic reaction centers from Rhodobacter sphaeroides

Picosecond transient circular dichroism spectra are reported for the primary intermediates in the photocycle of reaction centers isolated from Rhodobacter sphaeroides. The time-resolved circular dichroism spectra of the two electron transfer intermediates (BChl2) +BPh-LQA and (BChl2) +BPhLQ-A reveal a large, nonconservative, and fairly stationary CD band at 800 nm. These results suggests that mechanisms other than exciton interactions need to be included in order to explain the optical activity of this biological system.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P(Zn)), the accessory Zn-BChl a (B(Zn)), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P(Zn)(*), measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B(Zn)(*) to P(Zn), and the latter, the electron transfer from P(Zn) to H. The angle between the transition dipoles of B(Zn) and P(Zn) was estimated to be 36 degrees based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P(Zn).     

Interaction of photosynthetic pigments with various organic solvents 2. Application of magnetic circular dichroism to bacteriochlorophyll a and light-harvesting complex 1

Magnetic circular dichroism (MCD) and absorption spectra of metal bacteriochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q(y)(0-0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q(x)(0-0), and the ratio of MCD Q(y)(0-0) intensity to the dipole strength (B/D) was inversely proportional to the difference in energy between the Q(x)(0-0) and Q(y)(0-0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl(4)), however, the correlation did not hold because of the existence of aggregates. Hence, the correlation between the values of B/D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl beta-D-glucopyranoside.     

A BchD (magnesium chelatase) mutant of rhodobacter sphaeroides synthesizes zinc bacteriochlorophyll through novel zinc-containing intermediates

Heme and bacteriochlorophyll a (BChl) biosyntheses share the same pathway to protoporphyrin IX, which then branches as follows. Fe(2+) chelation into the macrocycle by ferrochelatase results in heme formation, and Mg(2+) addition by Mg-chelatase commits the porphyrin to BChl synthesis. It was recently discovered that a bchD (Mg-chelatase) mutant of Rhodobacter sphaeroides produces an alternative BChl in which Mg(2+) is substituted by Zn(2+). Zn-BChl has been found in only one other organism before, the acidophilic Acidiphilium rubrum. Our objectives in this work on the bchD mutant were to 1) elucidate the Zn-BChl biosynthetic pathway in this organism and 2) understand causes for the low amounts of Zn-BChl produced. The bchD mutant was found to contain a Zn-protoporphyrin IX pool, analogous to the Mg-protoporphyrin IX pool found in the wild type strain. Inhibition of ferrochelatase with N-methylprotoporphyrin IX caused Zn-protoporphyrin IX and Zn-BChl levels to decline by 80-90% in the bchD mutant, whereas in the wild type strain, Mg-protoporphyrin IX and Mg-BChl levels increased by 170-240%. Two early metabolites of the Zn-BChl pathway were isolated from the bchD mutant and identified as Zn-protoporphyrin IX monomethyl ester and divinyl-Zn-protochlorophyllide. Our data support a model in which ferrochelatase synthesizes Zn-protoporphyrin IX, and this metabolite is acted on by enzymes of the BChl pathway to produce Zn-BChl. Finally, the low amounts of Zn-BChl in the bchD mutant may be due, at least in part, to a bottleneck upstream of the step where divinyl-Zn-protochlorophyllide is converted to monovinyl-Zn-protochlorophyllide.     

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D⁺ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone-iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.     

Triplet-minus-singlet absorbance difference spectra of reaction centers and antenna pigments of the green photosynthetic bacterium Prosthecochloris aestuarii

We have applied absorbance-detected electron spin resonance in zero magnetic field to several pigment-protein complexes that belong to the membrane-bound photosystem of the green sulfur bacterium Prosthecochloris aestuarii. It was found that three triplet states can be discerned, that are formed in the light-harvesting bacteriochlorophyll a protein, the core complex and in the primary donor P-840, respectively. Triplet-minus-singlet absorbance difference spectra of the latter two states are presented. The spectrum of the core complex shows a bleaching at 837 nm and an absorbance increase at 808 nm. This suggests a strong electronic interaction between at least two of the constituent BChl a molecules of the complex. The triplet-minus-singlet spectrum of P-840 shows two negative bands at 826 and 837 nm, that, according to their linear dichroism, have almost parallel polarization. It is shown that no spectral evidence exists for the presence of two resolved dimer exciton bands of P-840. We conclude that P-840 either consists of two weakly coupled BChl a molecules or of a strongly coupled pair with one allowed exciton band at 837 nm, the other blue-shifted exciton component being very weak. Decay rates of P^T-840 of 6790 (±500) s⁻¹, 3920 (±300) s⁻¹ and 1275 (±100) s⁻¹ were observed for the x, y and z triplet sublevels, respectively.     

Analysis by exciton theory of the optical properties of the Chloroflexus aurantiacus reaction center

The optical properties of the reaction center of the filamentous green bacterium Chloroflexus aurantiacus, that contains three bacteriochlorophyll (BChl) a and three bacteriopheophytin (BPh) a molecules, were analyzed in the near-infrared region with the aid of exciton theory. The coordinates obtained from the X-ray analysis of the reaction center of Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) were used for the geometry of the reaction center of C. aurantiacus, with the replacement of one of the ‘accessory’ BChl molecules by BPh. The results were found to be in good agreement with experimental low-temperature absorption spectra, linear and circular dichroism and fluorescence polarization spectra and lead to the following conclusions. The allowed, low-energy exciton transition of the primary electron donor (P-865) is located at 887 nm and carries the dipole strength of approx. two BChl a monomers; the high-energy exciton transition, around 790 nm, is mixed with wave functions of other pigments, which explains its relatively small angle with respect to the 887 nm transition. The optical transition of the accessory BChl a molecule near 812 nm has some contribution of the BChls that constitute P-865. This can account for the experimentally observed reorientation and shift of this transition upon oxidation of P-865. Two of the BPh molecules are located on the same (probably the M) polypeptide subunit and show a clear splitting of absorption bands (11 nm) due to exciton coupling; the single BPh on the opposite branch shows hardly any exciton shift. Similar calculations for reaction centers of purple bacteria that contain four BChl a and two BPh a molecules resulted in a very low dipole strength for the high-energy transition of the primary donor due to antisymmetric mixing with both accessory BChl a wave functions and gave very little splitting of the absorption bands of BPh a. Our results indicate that the arrangement of the chromophores in reaction centers of C. aurantiacus is very similar to that in purple bacteria. The functional L-chains of the reaction centers of purple and filamentous green bacteria consist of pigments of the same type in a probably very similar arrangement.     

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800–B850 gap from 750 cm^\s-1 at 0.1 MPa to 900 cm^-1 at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800–850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800–B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C₉-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (-0.4 cm⁰¹/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm⁰¹/MPa). The shift rate for B850 at 4.2 K is-0.28 cm^–1/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.Abbreviations B800 BChl antenna band absorbing (at room temperature) at 800 nm (B850, B875, B1015 are defined similarly) - CD circular dichroism - FC factor Franck-Condon factor - FMO comple Fenna-Matthews-Olson complex - L-S theory Laird-Skinner theory - LH1 core light-harvesting complex of the BChl antenna complexes - LH2 peripheral light-harvesting complex of the BChl antenna complexes - NPHB non-photochemical hole burning - P960 absorption band of special pair of Rhodopseudomonas viridis absorbing at 960 nm (room temperature). P870 of Rhodobacter sphaeroides is defined similarly - QM/MM results quantum mechanical/molecular mechanical results - RC reaction center - ZPH zero phonon hole     

Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.     

Optically detected magnetic resonance of intact membranes from Chloroflexus aurantiacus. Evidence for exciton interaction between the RC and the B808–866 complex

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808–866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808–866 antenna complex. This evidence was provided by the comparison of the T–S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.This revised version was published online in October 2005 with corrections to the Cover Date.     

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes

The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Probing the bacteriochlorophyll binding site by reconstitution of the light-harvesting complex of Rhodospirillum rubrum with bacteriochlorophyll a analogues

Structural features of bacteriochlorophyll (BChl) a that are required for binding to the light-harvesting proteins of Rhodospirillum rubrum were determined by testing for reconstitution of the B873 or B820 (structural subunit of B873) light-harvesting complexes with BChl a analogues. The results indicate that the binding site is very specific; of the analogues tested, only derivatives of BChl a with ethyl, phytyl, and geranylgeranyl esterifying alcohols and BChl b (phytyl) successfully reconstituted to form B820- and B873-type complexes. BChl analogues lacking magnesium, the C-3 acetyl group, or the C-13(2) carbomethoxy group did not reconstitute to form B820 or B873. Also unreactive were 13(2)-hydroxyBChl a and 3-acetylchlorophyll a. Competition experiments showed that several of these nonreconstituting analogues significantly slowed BChl a binding to form B820 and blocked BChl a-B873 formation, indicating that the analogues may competitively bind to the protein even though they do not form red-shifted complexes. With the R. rubrum polypeptides, BChl b formed complexes that were further red-shifted than those of BChl a; however, the energies of the red shifts, binding behavior, and circular dichroism (CD) spectra were similar. B873 complexes reconstituted with the geranylgeranyl BChl a derivative, which contains the native esterifying alcohol for R. rubrum, showed in-vivo-like CD features, but the phytyl and ethyl B873 complexes showed inverted CD features in the near infrared. The B820 complex with the ethyl derivative was about 30-fold less stable than the two longer esterifying alcohol derivatives, but all formed stable B873 complexes.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.     

Picosecond-photodichroism studies of the transient states in Rhodopseudomonas sphaeroides reaction centers at 5 K. Effects of electron transfer on the six bacteriochlorin pigments

We have examined the dichroism of the visible and near-infrared absorption changes due to the early transient states in Rhodopseudomonas sphaeroides reaction centers imbedded in polyvinyl alcohol films at 5 K. The transient-state, ground-state and derivative spectra acquired under these conditions are highly resolved. Spectral features have been assigned to the bacteriochlorophyll (BChl) dimer (P) that serves as the primary electron donor, to each of the two additional BChls, and to the two bacteriopheophytin (BPh) molecules. The dichroism of the absorption changes, taken together with earlier results including our observation of a detection-wavelength dependence of the kinetics, argues that only one of the BPhs is a clearly resolved electron carrier prior to ubiquinone. The second BPh and the two BChls not constituting P display electrochromic effects and/or nuclear relaxations, possibly involving the protein, in response to the charge-separation process.     

Analysis of absorption spectra of purple bacterial reaction centers in the near infrared region by higher order derivative spectroscopy

Reaction centers (RCs) of purple bacteria are uniquely suited objects to study the mechanisms of the photosynthetic conversion of light energy into chemical energy. A recently introduced method of higher order derivative spectroscopy [I.K. Mikhailyuk, H. Lokstein, A.P. Razjivin, A method of spectral subband decomposition by simultaneous fitting the initial spectrum and a set of its derivatives, J. Biochem. Biophys. Methods 63 (2005) 10-23] was used to analyze the NIR absorption spectra of RC preparations from Rhodobacter (R.) sphaeroides strain 2R and Blastochloris (B.) viridis strain KH, containing bacteriochlorophyll (BChl) a and b, respectively. Q(y) bands of individual RC porphyrin components (BChls and bacteriopheophytins, BPheo) were identified. The results indicate that the upper exciton level P(y+) of the photo-active BChl dimer in RCs of R. sphaeroides has an absorption maximum of 810nm. The blue shift of a complex integral band at approximately 800nm upon oxidation of the RC is caused primarily by bleaching of P(y+), rather than by an electrochromic shift of the absorption band(s) of the monomeric BChls. Likewise, the disappearance of a band peaking at 842nm upon oxidation of RCs from B. viridis indicates that this band has to be assigned to P(y+). A blue shift of an absorption band at approximately 830nm upon oxidation of RCs of B. viridis is also essentially caused by the disappearance of P(y+), rather than by an electrochromic shift of the absorption bands of monomeric BChls. Absorption maxima of the monomeric BChls, B(B) and B(A) are at 802 and 797nm, respectively, in RCs of R. sphaeroides at room temperature. BPheo co-factors H(B) and H(A) peak at 748 and 758nm, respectively, at room temperature. For B. viridis RCs the spectral positions of H(B) and H(A) were found to be 796 and 816nm, respectively, at room temperature.     

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) 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 46nm 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? 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.     

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.