Electron transfer in reaction center core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum

Electron transfer in reaction center core (RCC) complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum was studied by measuring flash-induced absorbance changes. The first preparation contained approximately three iron-sulfur centers, indicating that the three putative electron acceptors F(X), F(A), and F(B) were present; the Chl. tepidum complex contained on the average only one. In the RCC complex of Ptc. aestuarii at 277 K essentially all of the oxidized primary donor (P840(+)) created by a flash was rereduced in several seconds by N-methylphenazonium methosulfate. In RCC complexes of Chl. tepidum two decay components, one of 0.7 ms and a smaller one of about 2 s, with identical absorbance difference spectra were observed. The fast component might be due to a back reaction of P840(+) with a reduced electron acceptor, in agreement with the notion that the terminal electron acceptors, F(A) and F(B), were lost in most of the Chl. tepidum complexes. In both complexes the terminal electron acceptor (F(A) or F(B)) could be reduced by dithionite, yielding a back reaction of 170 ms with P840(+). At 10 K in the RCC complexes of both species P840(+) was rereduced in 40 ms, presumably by a back reaction with F(X)(-). In addition, a 350 micros component occurred that can be ascribed to decay of the triplet of P840, formed in part of the complexes. For P840(+) rereduction a pronounced temperature dependence was observed, indicating that electron transfer is blocked after F(X) at temperatures below 200 K.     

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.     

Orientation and excitonic interactions of the Fenna—Matthews—Olson bacteriochlorophyll a protein in membranes of the green sulfur bacterium Chlorobium tepidum

Linear and circular dichroism spectra of isolated bacteriochlorophyll a proteins (FMO proteins) and membrane vesicles containing FMO protein from the green sulfur bacterium Chlorobium tepidum were measured at room temperature and 77 K. The orientation of membranes and isolated FMO protein was obtained by gel squeezing. Linear dichroism (LD) data indicate that isolated FMO protein and membrane vesicles associated with the FMO protein are oriented in a similar way in a squeezed polyacrylamide gel. Both samples show a characteristic negative LD band around 814 nm with flanking positive bands at 802 and 824 nm ascribed to the Q_y excitonic transitions of BChl a of the FMO protein. This confirms that the C3 symmetry axis of the trimer is perpendicular to the membrane plane, which is supported by the model of the disc-like structure of FMO protein trimers of Cb. tepidum [Li Yi-Fen, Zhou W, Blankenship RE, and Allen JP (1997) J Mol Biol 272: 456–471]. The LD data are consistent with either BChl 3 or 6, but not 7 as the principal contributor to the low temperature band at 825 nm. The low temperature linear and circular dichroism spectra of FMO protein trimers from Chlorobium tepidum show significant differences from the low temperature LD and CD spectra of FMO protein trimers from Prosthecochloris aestuarii. The data are interpreted in terms of somewhat different pigment-protein and pigment-pigment interactions in the two complexes.     

Pigment Composition in the Reaction Center Complex from the Thermophilic Green Sulfur Bacterium, Chlorobium tepidum: Carotenoid Glucoside Esters, Menaquinone and Chlorophylls

Distribution of pigments in the reaction center (RC) complex,chlorosomes and chlorosome-free membranes prepared from thegreen sulfur bacterium, Chlorobium tepidum, was analyzed. TheRC complex contained approximately 40 molecules of bacteriochlorophyll(BChl) a per P840, half of which are estimated to be in theFenna-Matthews-Olson (FMO) protein. Carotenes (2 molecules perP840) occupied only one third of the total carotenoids. Theremaining carotenoids (4 to 5 molecules per P840) were OH-chlorobacteneglucoside ester and OH-     

Time-Resolved Spectroscopy of Chlorophyll-a like Electron Acceptor in the Reaction Center Complex of the Green Sulfur Bacterium Chlorobium tepidum

The absorption changes of chlorophyll (Chl) a-like pigments(C670) were studied by ns-ms laser spectroscopy at 77 K in theuntreated and urea-treated homodimeric reaction center (RC)complex of the green sulfur bacterium Chlorobium tepidum. Theuntreated RC complex contained 9 molecules of C670 in additionto 41 molecules of Bchl a and 0.9 molecules of menaquinone-7per one primary electron donor Bchl a dimer (P840). Upon photo-oxidationof P840, C670 showed an absorption change of a red-shift withan isosbestic wavelength at 668 nm. The absorption change ofP840 decayed with time constants (t_1/e) of 55 and 37 ms at 283and 77 K, respectively, and was assigned to represent the chargerecombination between P840⁺ and FeS^–. In the urea-treatedRC complex, a bleach peaking at 670 nm with a shoulder peakat 662 nm, which is ascribable to the reduced primary electronacceptor A₀^–, was detected after the laser excitationin addition to the shift at 668 nm indicating the formationof the P840⁺A₀^– state. The P840⁺A₀^– state decayedwith a t_1/e of 43 ns at 77 K and produced a triplet state p840^Tdue to the suppression of the forward electron transfer. Theseresults indicate the two different types of C670 species inthe RC complex; the one peaking at 670 nm functions as A⁰, whilethe other peaking at 668 nm shows the electrochromic shift,which presumably functions as the accessory pigment locatedin the close vicinity of P840. (Received May 17, 1999; Accepted July 14, 1999)     

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.     

Electron transfer kinetics in purified reaction centers from the green sulfur bacterium Chlorobium tepidum studied by multiple-flash excitation.

Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.     

Energy transfer from carotenoid and FMO-protein in subcellular preparations from green sulfur bacteria. Spectroscopic characterization of an FMO-reaction center core complex at low temperature

The Fenna-Matthews-Olson (FMO)-protein and the FMO-reaction center (RC) core complex from the green sulfur bacterium Chlorobium tepidum were examined at 6 K by absorption and fluorescence spectroscopy. The absorption spectrum of the RC core complex was obtained by a subtraction method and found to have fiye peaks in the Q_Y region, at 797, 808, 818, 834 and 837 nm. The efficiency of energy transfer from carotenoid to bacteriochlorophyll a in the RC core complex was 23% at 6 K, and from the FMO-protein to the core it was 35%. Energy transfer from the FMO-protein to the core complex was also measured in isolated membranes of Prosthecochloris aestuarii from the action spectra of charge separation. Again, a low efficiency of energy transfer was obtained, both at 6 K and at room temperature.Abbreviations BChl- bacteriochlorophyll - P840- primary electron donor - RC- reaction center - FMO-protein- Fenna-Matthews-Olson-protein     

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.     

Spectral Irradiance and Distribution of Pigments in a Highly Layered Marine Microbial Mat

The spectral irradiance from 400 to 1,100 nm was measured with depth in the intertidal sand mats at Great Sippewissett Salt Marsh, Mass. These mats contained at least four distinct layers, composed of cyanobacteria, purple sulfur bacteria containing bacteriochlorophyll a (Bchl a), purple sulfur bacteria containing Bchl b, and green sulfur bacteria. Spectral irradiance was measured directly by layering sections of mat on a cosine receptor. Irradiance was also approximated by using a calibrated fiber-optic tip. With the tip, irradiance measurements could be obtained at depth intervals less than 250 μm. The irradiance spectra were correlated qualitatively and quantitatively with the distribution of the diverse chlorophyll pigments in this mat and were compared with spectra recorded in plain sand lacking pigmented phototrophs. We found that the shorter wavelengths (400 to 550 nm) were strongly attenuated in the top 2 mm of the mat. The longer wavelengths (red and near infrared) penetrated to much greater depths, where they were attenuated by Bchl a, b, and c-containing anoxygenic phototrophic bacteria. The specific attenuation bands in the irradiance spectra correlated with the specific in vivo absorption bands of the Bchl-protein complexes in the bacteria. We concluded that the pigments in the phototrophs had a profound affect on the light environment within the mat. It seems likely that the diverse Bchl-protein complexes found in the anoxygenic phototrophs evolved in dense mat environments as a result of competition for light.     

Nuclear wavepacket motion between P and P(+)B(A)(-) potential surfaces with subsequent electron transfer to H(A) in bacterial reaction centers. 1. Room temperature

Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P and of the charge transfer states P(+)B(A)(-) and P(+)H(A)(-) were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where B(A) and H(A) are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P at 935 nm, of the absorption band of B(A)(-) at 1020 nm, and of the bleaching band of H(A) at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm(-1) surface of P is directly induced by light absorption in P. When the wavepacket approaches the intersection between P and P(+)B(A)(-) surfaces at 120 and 380 fs delays, the formation of intermediate mixed-state emitting light at 935 nm (P) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the latter time, the wavepacket is transferred to the 32 cm(-1) mode which can belong to the P hypersurface effectively transferring the wavepacket to the P(+)B(A)(-) surface or can represent a diabatic surface which is formed by the states P and P(+)B(A)(-). The wavepacket motion on the P(+)B(A)(-) surface or on the P(+)B(A)(-) part of the mixing surface is accompanied by irreversible electron transfer to H(A). This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm(-1) mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm(-1) to the 32 cm(-1) mode is discussed.     

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.     

Low-temperature spectroscopy of isolated FMO-protein and a membrane-free reaction center complex from the green sulfur bacteriumChlorobium tepidum

We have isolated the water-soluble BChla-protein (FMO-protein) from the greer sulfur bacteriumChlorobium tepidum by a new procedure involving a salt-wash of isolated membranes at alkaline pH. The absorption spectrum of the isolated FMO-protein at 77 K was compared with that of a reaction-center complex containing the FMO-protein (FMO-RC complex) isolated fromC. tepidum following the procedure of Feiler U, Nitsche W and Michel H (1992) Biochemistry 31: 2608–2614. Oxidation or illumination of the FMO-RC complex caused bleaching of a component with a maximum at 836 nm which was not present in the purified FMO-protein.     

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.     

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium

The photosynthetic reaction center complex (RCC) of green sulfur bacteria (GSB) consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson (FMO). Functionally, FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs. In vivo, one RC was found to bind two FMOs, however, the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified. Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9 Å resolution by cryo-electron microscopy. The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously. We also observed two new subunits (PscE and PscF) and the N-terminal transmembrane domain of a cytochrome-containing subunit (PscC) in the structure. These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex. A new bacteriochlorophyll (numbered as 816) was identified at the interspace between PscF and PscA-1, causing an asymmetrical energy transfer from the two FMO trimers to RC core. Based on the structure, we propose an energy transfer network within this photosynthetic apparatus.     

Isolation and characterization of homodimeric type-I reaction center complex from Candidatus Chloracidobacterium thermophilum, an aerobic chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ～483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.     

Exciton dynamics in the chlorosomal antennae of the green bacteria Chloroflexus aurantiacus and Chlorobium tepidum

The energy transfer processes in isolated chlorosomes from green bacteria Chlorobium tepidum and Chloroflexus aurantiacus have been studied at low temperatures (1.27 K) by two-pulse photon echo and one-color transient absorption techniques with approximately 100 fs resolution. The decay of the coherence in both types of chlorosomes is characterized by four different dephasing times stretching from approximately 100 fs up to 300 ps. The fastest component reflects dephasing that is due to interaction of bacteriochlorophylls with the phonon bath, whereas the other components correspond to dephasing due to different energy transfer processes such as distribution of excitation along the rod-like aggregates, energy exchange between different rods in the chlorosome, and energy transfer to the base plate. As a basis for the interpretation of the excitation dephasing and energy transfer pathways, a superlattice-like structural model is proposed based on recent experimental data and computer modeling of the Bchl c aggregates (1994. Photosynth. Res. 41:225-233.) This model predicts a fine structure of the Q(y) absorption band that is fully supported by the present photon echo data.     

Specific inhibition of antenna bacteriochlorophyll synthesis in Chlorobium vibrioforme by anesthetic gases.

The green sulfur bacterium Chlorobium vibrioforme contains two types of bacteriochlorophyll (Bchl). The minor pigment, Bchl a, is associated primarily with the cell membrane and its reaction centers; and the major light-harvesting antenna pigment, Bchl d, is found primarily in the chlorosomes, which are attached to the inner surface of the cell membrane. Anesthetic gases, such as N2O, ethylene, and acetylene, were found to inhibit the synthesis of Bchl d, but not of Bchl a, thus allowing the cells to grow at high light intensities with a greatly diminished content of antenna pigment. Chlorosomes were absent or sparse in inhibited cells. Porphyrins accumulated in the inhibited cells. The major one was identified as the Bchl precursor magnesium-protoporphyrin IX monomethyl ester (Mg-PPME) by comparative absorption and fluorescence spectroscopy and thin-layer chromatography of the porphyrin and its derivatives with those of authentic protoporphyrin IX. Small amounts of Mg-PPME were present in control cells, but the addition of inhibitor caused a rapid increase in the Mg-PPME concentration, accompanying the inhibition of Bchl d synthesis. Cells grown in the presence of ethephon (as a source of ethylene) and allowed to stand in dim light for long periods accumulated large amounts of PPME and other porphyrins and excreted or released porphyrins, which accumulated as a brown precipitate in the culture. Inhibition of Bchl d synthesis was relieved upon removal of the inhibitor. These results suggest that the gases act at a step in pigment biosynthesis that affects the utilization of Mg-PPME for isocyclic ring formation. Synthesis of Bchl d and Bchl a may be differentially affected by the gases because of compartmentation of their biosynthetic apparatus or because competition for precursors favors Bchl a synthesis. An ethephon-resistant mutant strain was isolated by selection for growth in dim, long-wavelength light. The mutant cells were also resistant to acetylene, but not to N2O. The ability to reversibly generate viable Chlorobium cells that lack antenna pigments may be useful in photosynthesis research. The ethephon- and acetylene-resistant strain may be useful in the study of the enzymes and genes that are involved in the biosynthetic step that the gases affect.     

A membrane-bound monoheme cytochrome c551 of a novel type is the immediate electron donor to P840 of the Chlorobium vibrioforme photosynthetic reaction center complex.

A photosynthetic reaction center complex has been isolated from the green sulfur bacterium Chlorobium vibrioforme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 18, 15, 9, and 6 kDa. Only the 18-kDa polypeptide is stained with 3,3',5,5'-tetramethylbenzidine, a heme-specific reagent. Oxidized minus reduced difference spectra show the presence of approximately one heme/P840 and the presence of a cytochrome c551. Flash photolysis of P840 was followed by rereduction of P840+ and oxidation of cytochrome c551, both with a biphasic kinetic with t1/2 values of 7 and 50 microseconds. Using oligonucleotide probes derived from an N-terminal amino acid sequence of the 18-kDa polypeptide, a genomic clone was isolated. The sequence of the gene, which we designate cycA, predicts a single heme binding site (Cys-Asn-Lys-Cys-His). The 621-base pair open reading frame encodes an apoprotein of 22,858 Da with three predicted membrane-spanning alpha-helices. No extensive sequence similarity is found to other cytochromes. Northern blotting indicates that the cycA gene is transcribed as a monocistronic mRNA. Southern blotting shows the presence of only one cycA gene in the C. vibrioforme and Chlorobium tepidum genomes. The unique membrane-bound monoheme cytochrome c551 of C. vibriforme is assigned to a new class of c-type cytochromes. The implications for the current view of evolution of photosynthetic reaction center complexes are discussed.     

The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor for the F(X) iron-sulfur cluster in Chlorobium tepidum

The photosynthetic reaction center (RC) of green sulfur bacteria contains two [4Fe-4S] clusters named F(A) and F(B), by analogy with photosystem I (PS I). PS I also contains an interpolypeptide [4Fe-4S] cluster named F(X); however, spectroscopic evidence for an analogous iron-sulfur cluster in green sulfur bacteria remains equivocal. To minimize oxidative damage to the iron-sulfur clusters, we studied the sensitivity of F(A) and F(B) to molecular oxygen in whole cells of Chlorobium vibrioforme and Chlorobium tepidum and obtained highly photoactive membranes and RCs from Cb. tepidum by adjusting isolation conditions to maximize the amplitude of the F(A)(-)/F(B)(-) electron paramagnetic resonance signal at g = 1.89 (measured at 126 mW of microwave power and 14 K) relative to the P840(+) signal at g = 2.0028 (measured at 800 microW of microwave power and 14 K). In these optimized preparations we were able to differentiate F(X)(-) from F(A)(-)/F(B)(-) by their different relaxation properties. At temperatures between 4 and 9 K, isolated membranes and RCs of Cb. tepidum show a broad peak at g = 2.12 and a prominent high-field trough at g = 1.76 (measured at 126 mW of microwave power). The complete g-tensor of F(X)(-), extracted by numerical simulation, yields principal values of 2.17, 1.92, and 1. 77 and is similar to F(X) in PS I. An important difference from PS I is that because the bound cytochrome is available as a fast electron donor in Chlorobium, it is not necessary to prereduce F(A) and F(B) to photoaccumulate F(X)(-).