Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

Kinetics of electron transfer between soluble cytochrome c-554 and purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Soluble cytochrome c-554 (M _r 10 kDa) is purified from the green sulfur bacterium Chlorobium tepidum. Its midpoint redox potential is determined to be +148 mV from redox titration at pH 7.0. The kinetics of cytochrome c-554 oxidation by a purified reaction center complex from the same organism were studied by flash absorption spectroscopy at room temperature, and the results indicate that the reaction partner of cytochrome c-554 is cytochrome c-551 bound to the reaction center rather than the primary donor P840. The second-order rate constant for the electron donation from cytochrome c-554 to cytochrome c-551 was estimated to be 1.7×10⁷ M^–1 s^–1. The reaction rate was not significantly influenced by the ionic strength of the reaction medium.This revised version was published online in October 2005 with corrections to the Cover Date.     

Electron donation from membrane-bound cytochrome c to the photosynthetic reaction center in whole cells and isolated membranes of Heliobacterium gestii

The reaction between membrane-bound cytochrome c and the reaction center bacteriochlorophyll g dimer P798 was studied in the whole cells and isolated membranes of Heliobacterium gestii. In the whole cells, the flash-oxidized P798⁺ was rereduced in multiple exponential phases with half times (t _1/2s) of 10 s, 300 s and 4 ms in relative amplitudes of 40, 35 and 25%, respectively. The faster two phases were in parallel with the oxidation of cytochrome c. In isolated membranes, a significantly slow oxidation of the membrane-bound cytochrome c was detected with t _1/2 = 3 ms. This slow rate, however, again became faster with the addition of Mg²⁺. The rate showed a high temperature dependency giving apparent activation energies of 88.2 and 58.9 kJ/mol in the whole cells and isolated membranes, respectively. Therefore, membrane-bound cytochrome c donates electrons to the P798⁺ in a collisional reaction mode like the reaction of water-soluble proteins. The rereduction of the oxidized cytochrome c was suppressed by the addition of stigmatellin both in the whole cells and isolated membranes. This indicates that the electron transfer from the cytochrome bc complex to the photooxidized P798⁺ is mediated by the membrane-bound cytochrome c. The multiple flash excitation study showed that 2–3 hemes c were connected to the P798. By the heme staining after the SDS-PAGE analysis of the membraneous proteins, two cytochromes c were detected on the gel indicating apparent molecular masses of 17 and 30 kDa, respectively. The situation resembles the case in green sulfur bacteria, that is, the membrane-bound cyotochrome c _z couples electron transfer between the cytochrome bc complex and the P840 reaction center complex.This revised version was published online in October 2005 with corrections to the Cover Date.     

Orientation of reaction center complexes fromRhodobacter sphaeroides in proteoliposomes and the effect ofo-phenanthroline on electrogenesis during primary photochemical reaction

The orientation ofRhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown thato-phenanthroline, an inhibitor of electron transfer between primary (Q_A) and secondary (Q_B) quinone acceptors, can also inhibit the photoinduced Q_A reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-inducedo-phenanthroline inhibition of primary dipole (P-870⁺ · Q _A ^– ) formation is of a competitive nature.     

Genes encoding light-harvesting and reaction center proteins from Chromatium vinosum

Sequencing of a 3.4 kb DNA fragment isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum and of PCR products has resulted in identification of the Chr. vinosum pufL, pufM, and pufC genes, reading from the 5 to the 3 direction, and coding, respectively, for the L, M and cytochrome c subunits of the reaction center of this bacterium. Other PCR products have been used to obtain complete sequences for the pufB and pufA genes, located immediately upstream from pufL and encoding the apoproteins of two Chr. vinosum light- harvesting proteins. The 3-portion of the bchZ gene, a gene that codes for a protein involved in the biosynthesis of bacteriochlorophyll, has been located immediately upstream from pufB. A second pufB gene, pufB2, has been located downstream from pufC, as has the 5-portion of a second pufA gene, pufA2. The location of a second set of pufB and pufA genes, encoding light- harvesting proteins, downstream from pufC has not previously been reported for any photosynthetic bacterium. Translation of the gene sequences encoding these Chr. vinosum light-harvesting proteins reveals both similarities to and differences from the amino acid sequences, obtained from direct sequencing of the apoproteins, previously reported for Chr. vinosum light-harvesting proteins. Translation of these gene sequences, and of those for pufL, pufM and pufC, revealed significant homology, at the amino acid level, to the corresponding peptides of photosynthetic purple non-sulfur bacteria.     

Isolation and purification of membrane-bound cytochrome b-560 from photosynthetic bacterium Chromatium vinosum

A membrane-bound cytochrome of the b-type (cytochrome b-560) was success-fully purified from chromatophores of the photosynthetic purple sulfur bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate, sodium thiocyanate, and bacterial alkaline protease (EC 3·4·21·14) followed by gel filtration.The purified cytochrome b-560 showed the absorption maxima at 279, 412.5 and 533 nm in the oxidized form, and 427, 530 and 560 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 14.0 for a wavelength pair, 560 minus 540 nm.Isolated cytochrome b-560 was electrophoretically homogeneous, and its minimal molecular weight was estimated to the 13,000 by SDS polyacrylamide gel electrophoresis.The midpoint potential at pH 8.0 was –110mV, and was not dependent on the ambient pH in the pH range of 6.8 to 8.8.     

Oxidation of cytochrome c 2 by photosynthetic reaction centers of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in vivo. Effect of viscosity on the rate of reaction

In Rhodospirillum rubrum and Rhodopseudomonas sphaeroides it is shown that the oxidation of cytochrome c ₂ involves a diffusion limited process. From analysis of the results it follows that the electron transfer probability must be very low. This is corroborated by in vitro studies using the isolated components.     

Temperature dependence of energy transfer from the long wavelength antenna BChl-896 to the reaction center in Rhodospirillum rubrum,Rhodobacter sphaeroides (w.t. and M21 mutant) from 77 to 177K,studied by picosecond absorption spectroscopy

Decay of the bacteriochlorophyll excited state was measured in membranes of the purple bacteria Rhodospirillum (R.) rubrum, Rhodobacter (Rb.) sphaeroides wild type and Rb. sphaeroides mutant M21 using low intensity picosecond absorption spectroscopy. The excitation and probing pulses were chosen in the far red wing of the long wavelength absorption band, such that predominantly the minor antenna species B896 was excited. The decay of B896 was studied between 77 and 177K under conditions that the traps were active. In all species the B896 excited state decay is almost temperature independent between 100 and 177K, and probably between 100 and 300 K. In this temperature range the decay rates for the various species are very similar and close to 40 ps. Below 100 K this rate remains temperature independent in Rb. sphaeroides w. t. and M21, while in R. rubrum a steep decrease sets in. An analysis of this data with the theory of nuclear tunneling indicates an activation energy for the final transfer step from B896 to the special pair of 70cm^-1 for R. rubrum and 30cm^-1 or less for Rb. sphaeroides.Abbreviations B880 and B896 the main and long wavelength bacteriochlorophyll's of the LH-1 antenna - RC reaction centre - P special pair in the RC     

Microscopic simulation of quantum dynamics and nuclear tunneling in bacterial reaction centers

The dispersed polaron version of the semiclassical trajectory approach is used to evaluate the quantum mechanical nuclear tunneling effects in the charge recombination reaction, P⁺Q^–PQ, in photosynthetic bacterial reaction centers, The cclculations are based on the crystallographic structure of reaction centers from Rhodopseudomonas viridis. They succeed in capturing the temperature dependence of the rate constant without using adjustable parameters. This provides the first example of a microscopic simulation of quantum mechanical nuclear tunneling in a biological system.Abbreviations P bacteriochlorophyll dimer - Q ubiquinone in Rhodobacter sphaeroides, menaquinone in Rhodopseudomonas viridis - Rps. Rhodopseudomonas - Rb. Rhodobacter - QCFF/PI quantum mechanical extension of the consistent force field to -electron     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis

The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P⁺, a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c ₅₅₈) and two low potential (Cyt c ₅₅₃), act as secondary electron donors to P⁺. Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c ₅₅₈ by P⁺ with a rate of 3.7×10⁶s^-1 (=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×10⁵s^-1 (=3.5 sec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with -band maxima at 559 nm (Cyt c ₅₅₉) and 556.5 nm (Cyt c ₅₅₆), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c ₅₅₆Cyt c ₅₅₉P⁺. At lower redox potentials, a low midpoint potential cytochrome, Cyt c ₅₅₃, is preferentially oxidized by P⁺ with a rate of 7×10⁶s^-1 (=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c ₅₅₉, and it is suggested that a likely arrangement for the four hemes is: c ₅₅₃ c ₅₅₆ c ₅₅₃ c ₅₅₉P.Abbreviations diaminodurene 2,3,5,6-tetramethyl-p-phenylenediamine - MOPS 3-[N-morpholino]-propane-sulfonic acid - PMS methyl phenazinium methosulfate - PES ethyl phenazinium ethosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TX-100 Triton X-100     

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH ₄ ⁺ . Activity of the Fe protin component of nitrogenase required both Mn²⁺ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.Abbreviation HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid     

Applications of spectral hole burning spectroscopies to antenna and reaction center complexes

The underlying principles of spectral hole burning spectroscopies and the theory for hole profiles are reviewed and illustrated with calculated spectra. The methodology by which the dependence of the overall hole profile on burn wavelength can be used to reveal the contributions from site inhomogeneous broadening and various homogeneous broadening contributions to the broad Q_y-absorption bands of cofactors is emphasized. Applications to the primary electron donor states of the reaction centers of purple bacteria and Photosystems I and II of green plants are discussed. The antenna (light harvesting) complexes considered include B800–B850 and B875 of Rhodobacter sphaeroides and the base-plate complex of Prosthecochloris aestuarii with particular attention being given to excitonic interactions and level structure. The data presented show that spectral hole burning is a generally applicable low temperature approach for the study of excited state electronic and vibrational (intramolecular, phonon) structures, structural heterogeneity and excited state lifetimes.William E. Catron Fellow.     

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

The cytochrome bc 1 complexes of photosynthetic purple bacteria

Complete nucleotide sequences are now available for the pet (fbc) operons coding for the three electron carrying protein subunits of the cytochrome bc ₁ complexes of four photosynthetic purple non-sulfur bacteria. It has been demonstrated that, although the complex from one of these bacteria may contain a fourth subunit, three subunit complexes appear to be fully functional. The ligands to the three hemes and the one [2Fe-2S] cluster in the complex have been identified and considerable progress has been made in mapping the two quinone-binding sites present in the complex, as well as the binding sites for quinone analog inhibitors. Hydropathy analyses and alkaline phosphatase fusion experiments have provided considerable insight into the likely folding pattern of the cytochrome b peptide of the complex and identification of the electrogenic steps associated with electron transport through the complex has allowed the orientation within the membrane of the electron-carrying groups of the complex to be modeled.     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

Structural comparison of cytochrome c2and cytochrome c6

Photosynthesis Research - This review compares the three-dimensional structures of the solublec-type cytochromes that functionally link membrane-bound energy transducingcomplexes in algal,...     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

Pigment exchange of Photosystem II reaction center by chlorophyll d

Pigment exchanges among photosystem reaction centers (RCs) are useful for the identification and functional analysis of chromophores in photosynthetic organisms. Pigment replacement within the spinach Photosystem II RC was performed with Chl d derived from the oxygenic alga Acaryochloris marina, using a protocol similar to that reported previously [Gall et al. (1998) FEBS Lett 434: 88–92] based on the incubation of reaction centers with an excess of other pigments. In this study, we analyzed Chl d-modified monomeric RC which was separated from Chl d-modified dimeric RC by size-exclusion chromatography. Based on the assumption of a constant ratio of two Pheo a molecules per RC, the number of Chl a molecules in Chl d-modified monomeric RCs was found to decrease from six to four. The absorption spectrum of the Chl d-modified monomeric RC at room temperature showed a large peak at 699.5 nm originating from Chl d and a small peak at 672.5 nm orignating from Chl a. Photoaccumulation of the Pheo a⁻ in Chl d-modified monomeric RC, in the presence of sodium dithionate and methyl viologen, did not differ significantly from that in control RC, showing that the Chl d-modified monomeric RC retains its charge separation activity and photochemically active Pheo a.