Photooxidized Bacteriochlorophyll Dimer Research Articles

X-Ray Structure Determination of Isocytochrome C2 from the Photosynthetic Bacterium Rhodobacter sphaeroides

In cytochrome c2-deficient strains of the photosynthetic bacterium, Rhodobacter sphaeroides, a related c-type cytochome, isocytochome c2 replaces cytochrome c2 as the secondary electron donor to the photo-oxidized bacteriochlorophyll dimer in the reaction center. We will describe the x-ray structure determination of isocytochrome c2 at 2.2 Å from Rb sphaeroides.Analysis of the x-ray diffraction data indicated the crystals belong to space group P3(2) with unit cell dimensions a=b=56.0 Å c=90.5 Å and are merohederally twinned.

Effect of Anions on the Binding and Oxidation of Divalent Manganese and Iron in Modified Bacterial Reaction Centers

The influence of different anions on the binding and oxidation of manganous and ferrous cations was studied in four mutants of bacterial reaction centers that can bind and oxidize these metal ions. Light-minus-dark difference optical and electron paramagnetic resonance spectroscopies were applied to monitor electron transfer from bound divalent metal ions to the photo-oxidized bacteriochlorophyll dimer in the presence of five different anions. At pH 7, bicarbonate was found to be the most effective for both manganese and iron binding, with dissociation constants around 1 μM in three of the mutants. The pH dependence of the dissociation constants for manganese revealed that only bicarbonate and acetate were able to facilitate the binding and oxidation of the metal ion between pH 6 and 8 where the tight binding in their absence could not otherwise be established. The data are consistent with two molecules of bicarbonate or one molecule of acetate binding to the metal binding site. For ferrous ion, the binding and oxidation was facilitated not only by bicarbonate and acetate, but also by citrate. Electron paramagnetic resonance spectra suggest differences in the arrangement of the iron ligands in the presence of the various anions.

The structure and function of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c2 (cyt c2) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c2 (cyt c2) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c2 is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.

Toxicity effects on anaerobic biodegradation: a case study

Electron donors to photo-oxidized reaction centers were studied in Rhodopseudomonas palustris and Rhodopseudomonas acidophila using whole cells, membrane preparations and reaction center-B880 complexes. In Rps. palustris, no hemes were tightly bound to the reaction center complex and cytochrome c2 was presumed to be the direct electron donor to the photo-oxidized bacteriochlorophyll dimer in whole cells. Cytochrome c2 was lost in the membrane preparation obtained after a French press disruption of cells and mammalian cytochrome c added externally was oxidized by illumination. On the other hand, in Rps. acidophila, the reaction center complex was tightly associated with four cytochrome hemes, two of which were identified as cytochrome c-553 (Em7 = 110 mV) and the other two as cytochrome c-555 (Em7 = 360 mV). When both cytochromes were reduced prior to illumination, cytochrome c-553 was rapidly oxidized by a flash. Cytochrome c-555 was the one oxidized by a flash when cytochrome c-553 was already oxidized before activation. Cytochrome c2 was presumed to be the electron donor to the photo-oxidized cytochrome c-555 in whole cells. In the membrane preparation, a rapid electron transfer was observed from externally added mammalian cytochrome c to photooxidized cytochrome c-555. A possible phylogenetic correlation between the absence of the tightly bound cytochromes and a long class of cytochrome c2 (Dickerson, R.E. (1980) Nature 283, 210–212) is discussed.

The influence of electrostatic interactions and intramolecular dynamics on electron transfer from the cytochrome subunit to the cation ?radical of the bacteriochlorophyll dimer in reaction centers from Rps. viridis

Interheme electrostatic interaction can explain the acceleration of the electron transfer (ET) rate from the highest potential heme (C38o) to the photooxidized bacteriochlorophyll dimer (P+) which takes place after the reduction of neighbouring heme(s) of the cytochrome subunit in the reaction center of Rps. viridis. The electrostatic interaction energies calculated for neighbouring hemes, 7.0 A apart (edge-to-edge), and for two high potential hemes, 21.5 A apart are found to be 0.110 eV and 0.040 eV respectively. The reorganisation energy of the C380-P+ transition of about 0.290±0.030 eV is calculated using the Marcus theory of electron tunneling. An empirical relation for the rate of ET is given. The low temperature restriction of the C380-+ transition is caused by an energetic inhibition which originates from an opposite shifting of the energy levels of C380 and P+ due to the freezing of protein dynamics and protein-bound water mobility. The freezing of the protein dynamics is revealed by the Mossbauer effect and correlates with the efficiency of the ET.

Crystallization and X-ray structure determination of cytochrome c2 from Rhodobacter sphaeroides in three crystal forms.

Cytochrome c(2) serves as the secondary electron donor that reduces the photo-oxidized bacteriochlorophyll dimer in photosynthetic bacteria. Cytochrome c(2) from Rhodobacter sphaeroides has been crystallized in three different forms. At high ionic strength, crystals of a hexagonal space group (P6(1)22) were obtained, while at low ionic strength, triclinic (P1) and tetragonal (P4(1)2(1)2) crystals were formed. The three-dimensional structures of the cytochrome in all three crystal forms have been determined by X-ray diffraction at resolutions of 2.20 A (hexagonal), 1.95 A, (triclinic) and 1.53 A (tetragonal). The most significant difference observed was the binding of an imidazole molecule to the iron atom of the heme group in the hexagonal structure. This binding displaces the sulfur atom of Met l00, which forms the axial ligand in the triclinic and tetragonal structures.

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 2. Quantitative kinetic analysis

The electron-transfer kinetics from the soluble cytochrome (cyt) c2 to the photooxidized reaction center (RC) was studied with proteins isolated from Rhodobacter (R.) sphaeroides. In addition to wild-type (WT) RC, RCs harboring site-directed mutations at residue L162 (L162F, -M, -L, -S, or -G) wree analyzed. The disappearance of the absorption band of the photooxidized primary donor P+ (at 1250 nm) and the alpha-band of cyt c2 (at 550 nm) were monitored. Under conditions of high equimolar RC and cyt c2 concentrations, the kinetics were very similar to those measured in intact cells (Farchaus et al., 1993). The fast component of the kinetics normally seen in WT was not observed in any of the mutants; the overall rereduction rates for the mutants depended on the amino acid substitution. Light intensity, viscosity, ionic strength, and RC/cyt c2 stoichiometry of the reaction mixture were varied to distinguish the contributions of association, reorientation, and electron-transfer reactions to the observed kinetics. In competition experiments, WTRC (L162Y) and the mutant RCL162L showed similar affinity for cyt c2, with a dissociation constant of kD = 10(-6) M. Mutants with an aliphatic substitution at position L162 displayed slower cyt c2-RC association and dissociation rates. Comparison of the major kinetic component of the P+ rereduction rates for the aliphatic substitutions to the aromatic substitution, L162F, revealed that the former were less affected by ionic strength and viscosity than the latter. The viscosity and ionic strength dependences noted for L162F were comparable to those seen for the slow kinetic component observed for the WT RC. The redox midpoint potential of the P/P+ couple was increased by 30 mV (L162F) to 50 mV (L162L, G) over the WT value, leading to differences in delta G not large enough to account for the drastic kinetic effects. Rather, the results suggested that the state(s) where cyt c2 is nonproductively bound to the RC dominated in the mutants. In the L162F mutant, it appeared that only the distribution between the bound cyt c2 states was affected, whereas for the mutants with aliphatic substitutions, a decreased reorientation rate had to be additionally assumed in order to explain the observations.

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 1. Site-directed mutagenesis and initial characterization.

Five site-directed mutants were engineered to substitute phenylalanine, serine, leucine, methionine, and glycine for tyrosine residue 162 of the pufL gene in Rhodobacter (R.) sphaeroides. Each of the mutations and the wild-type (WT) genes was expressed in the R. sphaeroides puf deletion strain PUF delta LMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L162G and L162S were impaired. The amounts of mutant reaction centers expressed, the spectral characteristics, and the rates of intraprotein electron transfer and turnover were similar to the values obtained for WT. Kinetic measurements of photooxidized special pair rereduction mediated by the physiological donor cytochrome c2 in intact chemoheterotrophically grown cells revealed that the fast phase was abolished in all mutants and that the overall kinetics of rereduction was drastically slowed. It is concluded that L162Y plays a vital role in facilitating the rapid rereduction of the photooxidized bacteriochlorophyll dimer in R. sphaeroides.

Direct and indirect electron transfer from cytochromes c and c2 to the photosynthetic reaction center in pigment-protein complexes isolated from Rhodocyclus gelatinosus

Electron transfer from soluble cytochromes c and c 2 to the photo-oxidized bacteriochlorophyll dimer was studied in photosynthetic reaction center (RC) complexes and RC-B870 complexes isolated from Rhodocyclus gelatinosus. In the RC-B870 complex which consists of 6 polypeptides including a bound cytochrome subunit, cytochrome c from horse heart or cytochrome c 2, from R. gelatinosus was oxidized by photo-oxidized bacteriochlorophyll dimer indirectly via cytochrome c-555 in the complex. In the RC complex which is composed of two polypeptides, the oxidized bacteriochlorophyll dimer was reduced directly by the soluble cytochromes. Although direct electron transfer from soluble cytochrome does not function in R. gelatinosus in vivo, it would replace indirect electron donation if the bound cytochrome were lost mutationally.

Cytochromes functionally associated to photochemical reaction centers in Rhodopseudomonas palustris and Rhodopseudomonas acidophila

Electron donors to photo-oxidized reaction centers were studied in Rhodopseudomonas palustris and Rhodopseudomonas acidophila using whole cells, membrane preparations and reaction center-B880 complexes. In Rps. palustris, no hemes were tightly bound to the reaction center complex and cytochrome c 2 was presumed to be the direct electron donor to the photo-oxidized bacteriochlorophyll dimer in whole cells. Cytochrome c 2 was lost in the membrane preparation obtained after a French press disruption of cells and mammalian cytochrome c added externally was oxidized by illumination. On the other hand, in Rps. acidophila, the reaction center complex was tightly associated with four cytochrome hemes, two of which were identified as cytochrome c-553 ( E m7 = 110 mV) and the other two as cytochrome c-555 ( E m7 = 360 mV). When both cytochromes were reduced prior to illumination, cytochrome c-553 was rapidly oxidized by a flash. Cytochrome c-555 was the one oxidized by a flash when cytochrome c-553 was already oxidized before activation. Cytochrome c 2 was presumed to be the electron donor to the photo-oxidized cytochrome c-555 in whole cells. In the membrane preparation, a rapid electron transfer was observed from externally added mammalian cytochrome c to photooxidized cytochrome c-555. A possible phylogenetic correlation between the absence of the tightly bound cytochromes and a long class of cytochrome c 2 (Dickerson, R.E. (1980) Nature 283, 210–212) is discussed.

The reaction center and associated cytochromes of Thiocapsa pfennigii: Their thermodynamic and spectroscopic properties, and their possible location within the photosynthetic membrane

The majority of photosynthetic bacteria possess bacteriochlorophyll a, but two species, Rhodopseudomonas viridis and Thiocapsa pfennigii, possess bacteriochlorophyll b, which absorbs at significantly longer wavelengths. The reaction center of Rps. viridis has been extensively studied; this paper presents studies on the reaction center of T. pfennigii, with the following results. 1. (i) The “primary donor” bacteriochlorophyll dimer of T. pfennigii has a midpoint redox potential of +490 mV at pH 7, and the oxidized form has a gaussian EPR signal centered close to g = 2.0025 with a peak-to-peak width of 13 G. The zero field splitting characteristics of the light-induced triplet state of the bacteriochlorophyll dimer, seen when the “primary acceptor” is reduced before illumination, are D = 158 · 10 −4 cm −1, E = 39 · 10 −4 cm −1. These EPR properties are very similar to those of the bacteriochlorophyll dimer of Rps. viridis, and are quite distinct from those of bacteriochlorophyll a-containing species. 2. (ii) The intermediary carrier, I, which is probably a bacteriopheophytin b molecule in close association with the “primary acceptor”, can be trapped in its reduced form by the illumination of appropriately poised samples at 200 K. The EPR signals associated with I −· are very similar to those of Chromatium vinosum, with the majority of the spins being observed in the form of a broad signal centered close to g = 2.003, and split by 68 G; the magnitude of the splitting of this signal is only half that seen in Rps. viridis. We have been unable to measure the E m of the I/I −· couple in T. pfennigii. 3. (iii) The “primary acceptor” of T. pfennigii is very similar to that of the majority of other purple bacterial species, being characterized by an EPR signal at g = 1.82 and g = 1.62. The E m of the redox couple is pH-dependent below pH 6.5, and the E m of the unprotonated couple, which is believed to function in electron flow, is −130 mV. 4. (iv) Four cytochrome hemes can apparently donate electrons to the photooxidized bacteriochlorophyll dimer free radical cation, two of cytochrome c-555 ( E m7 = 340 mV) and two of cytochrome c-550 ( E m7 = 0). If all are reduced prior to illumination, the latter are preferentially oxidized. 5. (v) The carotenoid bandshift of T. pfennigii can be interpreted as indicating that the bacteriochlorophyll dimer is located near the middle of the membrane dielectric, with the high potential cytochrome c-555 closer to the inside, and quinone · iron closer to the outside of the chromatophore membrane. The low potential cytochrome c-550 seems to be nearer the outside of the membrane than the bacteriochlorophyll dimer.