EPR and optical spectroscopic properties of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum.

1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K. 2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are equivalent with respect to the light induced (BChl)2+. At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity. 3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow (t1/2 approximately equal to 30 s) oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinone-iron. The state (oxidized c553/reduced I) relaxes in the dark at 200K in t1/2 approx. 20 min but below 77 K it is trapped on a days time scale. 4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I-. is consistent with the idea that the reduced intermediary carrier is not simply BPh-. but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state...     

Aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c at low temperature

The effect of temperature on the aggregation of 3^lR-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of n-pentane and methylcyclohexane (1/1, v/v) was studied by means of absorption, circular dichroism and fluorescence spectroscopy. At room temperature essentially only two aggregate species, absorbing at 702 nm (A-702) and 719 nm (A-719), were present. Upon cooling to 219 K, A-702 was quantitatively converted to A-719. Further lowering of the temperature led to the stepwise formation of larger aggregates by the conversion of A-719 to aggregate species absorbing at 743 nm (A-743) and 755 nm (A-755). All absorption changes were reversible. A-719 was highly fluorescent (maximum at 192 K: 744 nm), while A-743 and especially A-755 were weakly fluorescent. Below 130 K the mixture solidified, and no major changes in the absorption spectrum were observed upon further cooling. At 45 K, however, a relatively strong emission at 775 nm was observed. Below 200 K, the absorption, fluorescence and circular dichroism spectra resembled that of the chlorosome. These results open up the possibility to study higher aggregates of BChl c as models for the chlorosome by various methods at low temperature, thus avoiding interference by thermal processes.Abbreviations A-680, A-702, A-719, A-743 and A-755- BChl c aggregates absorbing at the wavelengths indicated - BChl- bacteriochlorophyll - R[E,E] BChl c _F- the 3¹ R isomer of 8,12-diethyl BChl c esterified with farnesol (F), analogously - M- methyl - Pr- propyl - S- stearol (see Smith 1994) - CD- circular dichroism     

Titles of related papers in other sections

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1 : 2 : 2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846 and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b. The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably, to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P⁺-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.     

Primary photosynthetic processes in Heliobacterium chlorum at 15K

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Q_y region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.Abbreviations BChl bacteriochlorophyll - FWHM full width at half maximum - P-798 primary electron donor - Tris tris(hydroxymethyl)amino methane     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)₂, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)₂ in the first singlet excited state, (BChl)1₂, through the bacteriopheophytin, BPh, to the primary ubiquinone, Q_A. Possible contributions to the field-induced loss of quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation are: (1) a decrease in the free-energy gap between the states (BChl)1₂ and (BChl)^⨥₂BPh^⨪Q_A, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)^⨥₂BPh^⨪Q_A directly or via the (BChl)₂ triplet state to the ground state and (3) an impeded electron transfer from (BChl)^⨥₂BPh^⨪Q_A to (BChl)^⨥₂BPhQ^⨪_A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.     

Localization and concentration of hydroxylamine oxidoreductase and cytochromes c-552, c-554, cm-553, cm-552 and a in Nitrosomonas europaea

Two membrane-associated cytochromes, cytochrome c_m-553 and cytochrome c_m-552, were derived from Nitrosomonas europaea. The major c-type cytochrome, cytochrome c_m-553, accounted for 92% of the c heme found in the membrane. It had absorption maxima at 410 nm in the oxidized form and at 417, 523 and 553 nm in the dithionite reduced form. Cytochrome c_m-552 possessed absorption maxima at 409 nm in the oxidized form, at 421, 522 and 552 in the dithionite reduced form, and at 418 in the dithionite reduced plus CO form. The concentration and cellular distribution of the two c-type membrane cytochromes, hydroxylamine oxidoreductase and cytochromes c-552, c-554, and a were determined. Over 95% of the soluble cytochromes (hydroxylamine oxidoreductase cytochromes and c-552 and c-554) were periplasmic, whereas cytochrome c_m-553, cytochrome c_m-552 and cytochrome a were associated with the cell membrane. The outer membrane and cytoplasm were devoid of cytochromes. The extracytoplasmic location of the proton-yielding hydroxylamine oxidizing system (NH₂OH ™ HNO + 2H⁺ + 2e⁻) may contribute to an energy-linked proton gradient. The heme concentrations of hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 2.4, 1.2, 0.3, 1.3, 0.1 and 1.1 nmol/mg cell protein, respectively. The corresponding molar ratios of heme were 22:11:2.9:12:1.0:10. The enzyme or cytochrome concentrations for hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 0.13, 1.05, 0.09, 0.63, 0.055 and 0.56 nmol/mg cell protein, respectively. The corresponding molar ratios were 0.24:2.2:0.16:1.2:0.1:1.0.     

Manipulation of the bacteriochlorophyll c homolog distribution in the green sulfur bacterium Chlorobium tepidum

We have shown that the green sulfur bacterium Chlorobium tepidum can be grown in batch culture supplemented with potentially toxic fatty alcohols without a major effect on the growth rate if the concentration of the alcohols is kept low either by programmed addition or by adding the alcohol as an inclusion complex with -cyclodextrin. HPLC and GC analysis of pigment extracts from the supplemented cells showed that the fatty alcohols were incorporated into bacteriochlorophyll c as the esterifying alcohol. It was possible to change up to 43% of the naturally occurring farnesyl ester of bacteriochlorophyll c with the added alcohol. This change in the homolog composition had no effect on the spectral properties of the cells when farnesol was partially replaced by stearol, phytol or geranylgeraniol. However, with dodecanol we obtained a blue-shift of 6 nm of the Q_y band of the bacteriochlorophyll c and a concomitant change in the fluorescence emission was observed. The possible significance of these findings is discussed in the light of current ideas about bacteriochlorophyll organization in the chlorosomes.Abbreviations -CD -cyclodextrin - BChl bacteriochlorophyll - BChl c _H bacteriochlorophyllide c - [E,M] BChl c _F 8-ethyl, 12-methyl, farnesyl BChl c - [E,E] BChl c _F 8-ethyl, 12-ethyl, farnesyl BChl c - [P,E] BChl c _F 8-propyl, 12-ethyl, farnesyl BChl c - [I,E] BChl c _F 8-isobutyl, 12-ethyl, farnesyl BChl c - Car carotenoids     

The effect of detergent on the structure and composition of chlorosomes isolated from Chloroflexus aurantiacus

Isolated chlorosomes, treated with the detergent lithium dodecyl sulfate (LDS), can be separated into two green fractions by agarose gel electrophoresis. One fraction contains chlorosomes with a full complement of proteins and antenna BChl c absorbing at 740 nm, but with a more spherical form than the normal ellipsoid shape observed in control chlorosomes. The second fraction was completely devoid of proteins but had a similar absorption spectrum. Electron micrographs of the protein-free fraction indicated the presence of stain-excluding spheres with overall dimensions resembling those of intact chlorosomes (40–100 nm). These spheres are probably micelles of BChl c liberated from the chlorosomes during the detergent treatment, since similar structures could be produced when purified BChl c, dissolved in 1-hexanol, was dispersed in buffer, producing an aggregate absorbing at 742 nm. These results suggest that the chlorosome proteins are not required to produce an arrangement of BChl c chromophores which gives rise to a 740 nm absorption peak resembling that of intact chlorosomes. It seems probable, however, that proteins have a role in determining the overall shape of the chlorosome. Treatment with cross-linking reagents did not prevent the detergent-induced changes in chlorosome morphology.Abbreviations BChl bacteriochlorophyll - DSP dithiobis-succinimidyl-2-propionate - EM electron microscopy - LDS lithium dodecyl sulfate - MGDG monogalactosyl diacylglycerol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Hole burning study of excited state structure and energy transfer dynamics of bacteriochlorophyll c in chlorosomes of green sulphur photosynthetic bacteria

Results of low temperature fluorescence and spectral hole burning experiments with whole cells and isolated chlorosomes of the green sulfur bacterium Chlorobium limicola containing BChl c are reported. At least two spectral forms of BChl c (short-wavelength and long-wavelength absorbing BChl c) were identified in the second derivative fluorescence spectra. The widths of persistent holes burned in the fluorescence spectrum of BChl c are determined by excited state lifetimes due to fast energy transfer. Different excited state lifetimes for both BChl c forms were observed. A site distribution function of the lowest excited state of chlorosomal BChl c was revealed. The excited state lifetimes are strongly influenced by redox conditions of the solution. At anaerobic conditions the lifetime of 5.3 ps corresponds to the rate of energy transfer between BChl c clusters. This time shortens to 2.6 ps at aerobic conditions. The shortening may be caused by introducing a quencher. Spectral bands observed in the fluorescence of isolated chlorosomes were attributed to monomeric and lower state aggregates of BChl c. These forms are not functionally connected with the chlorosome.Abbreviations BChl bacteriochlorophyll - EET electronic energy transfer - FWHM full width at half maximum - SDF site distribution function - RC reaction centre     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Pigment composition of the photosynthetic membrane and reaction center of the green bacterium Prosthecochloris aestuarii

We have performed a quantitative analysis of the pigment composition of different pigment-protein complexes present in the membrane of the green sulfur bacterium Prosthecochloris aestuarii, using the resolving power of reversed-phase high-performance liquid chromatography. The most purified photochemically active complexes contained only carotenoids (OH-chlorobactene and rhodopin), bacteriochlorophyll a and a chlorophyllous pigment with absorption maxima at 663 and 433 nm, like bacteriochlorophyll c. However, the lipophilicity of this pigment, labeled BChl 663, is quite high and indicates that it contains 5–6 additional methylene groups compared to the BChl c homologue known as most lipophilic. Comparison of the BChl 663 content of various pigment-protein complexes indicates that BChl 663 is present in an amount of 10–15 molecules per reaction center. BChl 663 absorbs at 670 nm in vivo, with a specific extinction coefficient of 85 (±10) mM⁻¹ · cm⁻¹. In view of the evidence that the primary electron acceptor in P. aestuarii is a pigment with absorption maximum at 670 nm (Nuijs, A.M., Vasmel, H., Joppe, H.L.P., Duysens, L.N.M. and Amesz, J. (1985) Biochim. Biophys. Acta 807, 24–34) a direct consequence of these experiments is the fact that only BChl 663 can be a likely candidate for the role of primary electron acceptor as no other pigments absorbing around 670 nm (e.g., bacteriopheophytin c) are present in a photochemically active pigment-protein complex derived from the membrane of this green bacterium.     

The photochemical reaction center of the bacteriochlorophyll b-containing organism Thiocapsa pfennigii

A photochemical reaction-center preparation has been made from a second bacteriochlorophyll b-containing organism, Thiocapsa pfennigii. The reaction-center unit is thought to be composed of one P-960, four bacteriochlorophyll, two bacteriopheophytin, one carotenoid molecules and polypeptides of M_r 40000, 37000, 34000, 27000 and 26000 probably plus quinones and metal atoms. The preparation also contains a low-potential cytochrome c-555 and a high-potential cytochrome c-557 bound to the reaction center in a 3–4:2–3:1 molar ratio with respect to P-960. The 40 kDa subunit is associated with the cytochromes, while the 37, 34 and 27 + 26 kDa subunits are proposed to be equivalent to the H, M and L polypeptides of bacteriochlorophyll a-containing reaction centers. The cytochromes are oxidized by P-960⁺. The three near-infrared absorption bands at 788, 840 and 968 nm are assigned to bacteriopheophytin, bacteriochlorophyll and the primary donor (P-960), respectively. The 778 nm peak resolves into two at 77 K; no further resolution of the other two peaks occurs. Illumination of the sodium dithionite-reduced reaction centers at 77 K by 960 nm-light results in P-960, transferring one electron from cytochrome c-555 mainly to a bacteriopheophytin molecule, absorbing at 781 nm. A similar treatment at room temperatures reduces most of the two bacteriopheophytin molecules. It is argued that both bacteriopheophytin molecules, possibly with some contribution from bacteriochlorophyll, form an intermediary electron-carrier complex between P-960 and a quinone in T. pfennigii. We could not substantiate that a bacteriochlorophyll molecule precedes the bacteriopheophytins in the electron transfer sequence. Although the biochemical characteristics of the reaction center are very similar to those of the other known bacterioclorophyll b-containing reaction center, that from Rhodopseudomonas viridis, their spectral characteristics are not. This has helped elucidate more about the function of each spectral form and led us to conclude that the 850 nm form in Rps. viridis is not the higher energy transition of the special pair of bacteriochlorophyll molecules forming P-960. Laser-flash-in-duced absorbance changes in T. pfennigii reaction-center preparation should now lead to a more complete understanding of the mechanism of the primary photochemical event.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations

1. The reduced minus oxidized extinction coefficients (Δ^red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM⁻¹ · cm⁻¹ with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM⁻¹ · cm⁻¹ on dual wavelength mode set at 605 – 540 nm. Cytochrome c₂, which is not affected by detergent, has a Δ^red-ox value at 550-540 nm of 19.0 mM⁻¹ · cm⁻¹.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100 : 1. The cytochrome c₂: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c₂ molecules (intact P-c₂ units). The remaining reaction center proteins are not associated with cytochrome c₂ on a kinetically viable basis and may be the result of damage incurred during mechanical rupture of the cells.3. The half-reduction potential of cytochrome c₂ in the isolated state is 345 mV. In the chromatophore, two electrochemical species of cytochrome c₂ are recognized. The majority has a value of approx. 295 mV and is identifiable with cytochrome c₂ in a reaction center protein-associated state (kinetically active, intact P-c₂ units); the remainder has an approx. 350 mV half-reduction potential and is probably cytochrome c₂ in the “free” or reaction center-dissociated state (possibly from damaged P-c₂ units). It appears that there is no exchange of cytochrome c₂ between the reaction center-associated and the reaction center-dissociated state.4. The half-reduction potential of cytochrome c₂ is pH independent (from pH 5 to 9) whether measured in the free state or when associated with the chromatophore membrane. This shows that a proton is not involved in the oxidation and reduction of cytochrome c₂ in the physiological pH range.5. The kinetics of the intact reaction center, P, and cytochrome c₂ units in chromatophores and whole cells of Rhodopseudomonas spheroides are described. The two cytochrome c₂ molecules which are associated with one P exhibit similar oxidation kinetics; both are biphasic. The fast phase is estimated to be 20–40 μs in half time. The second slower phase is variable depending on the ionic strength of the medium used for the preparation of the chromatophores; it varies from 0.3 to 8 ms.6. An equilibrium for cytochrome c₂ and the reaction center and/or the membrane is suggested. The two states of the equilibrium are described by a population of cytochrome c₂ functionally “close” to the P⁺, and a population functionally distant from the P⁺, which might be physically off the binding site, or orientated unfavorably to the P⁺. The former population is identified by the 20–40 μs oxidation rate; the latter variable and somewhat slower oxidation (0.3–8 ms) is that whose rate is governed by the diffusional processes of the equilibrium which brings the cytochrome to the close position.7. Carotenoid bandshifts are kinetically compatible (a) with the P oxidation which is too fast to measure, and (b) with the two phases of cytochrome c₂ oxidation. These are interpreted as arising from local electric field alterations occurring during the electron transfer events in the reaction center and cytochrome c₂.     

Electron spin resonance characterization of Chromatium D hemes,non-heme irons and the components involved in primary photochemistry

The combination of redox potentiometry with low temperature electron spin resonance (ESR) spectroscopy has led to further characterization of electron transfer components of Chromatium D. These include the readily buffer-soluble cytochromes c₅₅₃ and c′ and the high-potential iron-sulfur protein in the isolated state and associated with the chromatophore membrane. Buffer-insoluble cytochrome c₅₅₃, cytochro—me c₅₅₅, bacteriochlorophyll and the primary electron acceptor have been characterized both in the chromatophore membrane and also in a sodium dodecylsulfate detergent-solubilized subchromatophore preparation. Two iron-sulfur proteins have been revealed which are present in the chromatophore membrane but are released on treatment with sodium dodecylsulfate. They have central g values at 1.90 and 1.94 and have estimated midpoint potentials at pH 7.4 (E_m7·4) at +280 mV and ?100 mV, respectively, when associated with the chromatophore.In the membrane associated state the apparent E_m of cytochrome c′ is approximately 200 mV more positive than the E_m values reported for the free state; this implies either that the reduced form of cytochrome c′ binds to the membrane (or to a component therein) to a degree which is > 10³ times greater than that of the oxidized form or that the E_m shift results from membrane solvation. In the case of the high-potential iron-sulfur protein however, its E_m when associated with the chromatophore membrane is similar to that reported in the isolated state. The light-induced oxidation of the high-potential iron-sulfur protein at room temperature appears to be linked only to the oxidation of cytochrome c₅₅₅; it could serve as an electron pool in equilibrium with cytochrome c₅₅₅ in the cyclic electron flow system.The redox component defined in the reduced state by its g_y = 1.82 and g_x = 1.62 ESR spectrum satisfies the following criteria for its identification as the primary electron acceptor of P883. (a) The E_m7·4 value of the g = 1.82 component is ?120 ± 25mV. (b) At ?70 mV, where the g = 1.82 component is mainly oxidized in the dark, brief illumination at low temperature which causes the irreversible oxidation of one cytochrome c₅₅₃ heme, also induces the permanent reduction of the g = 1.82 component; the extent of reduction after brief illumination, given by the g = 1.82 signal height, is the same as that induced chemically at ?270 mV showing it to be fully reduced by the receipt of a single electron. (c) At more positive potentials where cytochrome c₅₅₃ is oxidized and is not involved in low-temperature reactions, the light-induced low-temperature kinetics of the g = 1.82 signal are reversible; the flash-induced g = 1.82 formation and subsequent dark decay are the same as those for the flash-induced P⁺883 (g = 2) formation and dark decay. We suggest that until a full physical-chemical characterization is completed this g = 1.82 component be designated “photoredoxin”.