Bacteriopheophytin

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Neal W Woodbury - One of the best experts on this subject based on the ideXlab platform.

  • electron transfer in bacterial reaction centers with the photoactive Bacteriopheophytin replaced by a bacteriochlorophyll through coordinating ligand substitution
    Biochemistry, 2016
    Co-Authors: Rafael G Saer, Thomas J Beatty, Neal W Woodbury
    Abstract:

    The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the Bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a Bacteriopheophytin in the HA pocket, forming so-called β-type mutants (in which the HA cofactor is called βA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniqu...

  • the protein environment of the Bacteriopheophytin anion modulates charge separation and charge recombination in bacterial reaction centers
    Journal of Physical Chemistry B, 2013
    Co-Authors: Rafael G Saer, Thomas J Beatty, Neal W Woodbury
    Abstract:

    The kinetics and pathway of electron transfer has been explored in a series of reaction center mutants from Rhodobacter sphaeroides, in which the leucine residue at M214 near the Bacteriopheophytin cofactor in the A-branch has been replaced with methionine, cysteine, alanine, and glycine. These amino acids have substantially different volumes, both from each other and, except for methionine, from the native leucine. Though the mutation site of M214 is close to the Bacteriopheophytin cofactor, which is involved in the electron transfer, none of the mutations alter the cofactor composition of the reaction center and the primary charge separation reaction is essentially undisturbed. However, the kinetics of electron transfer from HA– → QA becomes both slower and substantially heterogeneous in three of the four mutants. The decreased HA– → QA electron transfer rate allows charge recombination between P+ and HA– to compete with the forward reaction, resulting in a drop in the overall yield of charge separation...

  • b side electron transfer in a rhodobacter sphaeroides reaction center mutant in which the b side monomer bacteriochlorophyll is replaced with Bacteriopheophytin low temperature study and energetics of charge separated states
    Journal of Physical Chemistry B, 2002
    Co-Authors: Evaldas Katilius, Zivile Katiliene, Aileen K W Taguchi, Neal W Woodbury
    Abstract:

    The mutation, HL(M182), in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll (BChl) on the inactive side (B side) of the RC with a Bacteriopheophytin (BPheo; the new cofactor is referred to as o B ). In o B -containing RCs, the first excited state of the primary donor (P * ) decays with an accelerated time constant of 2.6 ′ 0.1 ps at room temperature as compared to 3.1 ′ 0.2 ps in wild-type (WT) RCs. At low temperatures, P * decay is essentially the same in the HL(M182) mutant and WT RCs: 1.4 ′ 0.1 ps at 77 K and 1.1 ′ 0.1 ps at 9 K. The yield of the charge-separated P + o B - state decreases from 35% at room temperature to 12% at 77 and 9 K. The decreased P + o B - yield is apparently due to the fact that the rate of the charge separation along the A side of the RC at low temperature increases, while the rate along the B side remains essentially unchanged. From measurements of the long-lived fluorescence decay at room temperature, the standard free energy of the P + o B - state is estimated to be about 0.16 ′ 0.04 eV below P * . Given a difference between the midpoint potentials of BChl and BPheo of 0.26 ′ 0.03 V, the standard free energy of the P + B B - state in WT RC is estimated to be 0.1 ′ 0.07 eV above P * .

  • b side electron transfer in a rhodobacter sphaeroides reaction center mutant in which the b side monomer bacteriochlorophyll is replaced with Bacteriopheophytin
    Journal of Physical Chemistry B, 1999
    Co-Authors: Evaldas Katilius, Trieva Turanchik, And Aileen K W Taguchi, Neal W Woodbury
    Abstract:

    The mutation, H(M182)L, in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll on the inactive side (B-side) of the RC with a Bacteriopheophytin (the new cofactor is referred to as φB). In φB containing RCs, P* stimulated emission decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature compared to 3.1 ± 0.2 ps in WT RCs. Analysis of the time-resolved spectra implies that two states are being formed during the initial reaction in the mutant:  the usual P+HA- state, as seen in WT, and a new state, P+φB-. P+φB- is formed during the decay of P* and recombines to the ground state with a lifetime of 200 ± 20 ps. The yield of the P+φB- state is 35 ± 5% at room temperature, while the remaining 65 ± 5% of the initial electron-transfer results in P+HA-. There does not appear to be any further electron transfer from φB- to HB. Apparently, in the H(M182)L mutant, the state P+φB- is lower in free energy than the P+HB- state.

Christine Kirmaier - One of the best experts on this subject based on the ideXlab platform.

  • temperature dependence of electron transfer to the m side Bacteriopheophytin in rhodobacter capsulatus reaction centers
    Journal of Physical Chemistry B, 2008
    Co-Authors: Jessica I Chuang, Dewey Holten, Steven G Boxer, Christine Kirmaier
    Abstract:

    Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the DLL mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the DLL-FYLFM mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native Bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845−3851). For DLL, depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings...

  • high yield of m side electron transfer in mutants of rhodobacter capsulatus reaction centers lacking the l side Bacteriopheophytin
    Biochemistry, 2006
    Co-Authors: Jessica I Chuang, Dewey Holten, Steven G Boxer, Christine Kirmaier
    Abstract:

    We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatusDLL mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the DLL mutant in chromatophore preparations showed that RCs assembled without the Bacteriopheophytin HL electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D LL RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of HL. The excited state of the primary electron donor, P*, is found to have a lifetime of 180 ( 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the DLL background of two residues that affect the P/P + oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P + HM - , more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.

  • resonance raman characterization of reaction centers with an asp residue near the photoactive Bacteriopheophytin
    Biochemistry, 1998
    Co-Authors: Christine Kirmaier, Dewey Holten, David F Bocian
    Abstract:

    Qy-excitation resonance Raman (RR) studies are reported for a series of Rhodobacter capsulatus reaction centers (RCs) containing mutations at L-polypeptide residue 121 near the photoactive Bacteriopheophytin (BPhL). The studies focus on the electronic/structural perturbations of BPhL induced by replacing the native Phe with an Asp residue. Earlier work has shown that the electron-transfer properties of F(L121)D RCs are closely related to those of RCs in which BPhL is replaced by bacteriochlorophyll (BChl) (beta-type RCs) or by pheophytin. In addition to the F(L121)D single mutant, RR studies were performed on the F(L121)D/E(L104)L double mutant, which additionally removes the hydrogen bond between BPhL and the native Glu L104 residue. The vibrational signatures of BPhL in the single and double mutants containing Asp L121 are compared with one another and with those of BPhL in both wild-type and F(L121)L RCs. The replacement of the aromatic Phe residue with Leu has no discernible effect on the vibrational properties of BPhL, a finding in concert with the previously reported absence of an effect of the mutation on the electron-transfer characteristics of the RC. In contrast, replacement of Phe with Asp significantly perturbs the vibrational characteristics of BPhL, and in a manner most consistent with Asp L121 being deprotonated and negatively charged. The negative charge of the carboxyl group of Asp L121 interacts with the pi-electron system of BPhL in a relatively nonspecific fashion, diminishing the contribution of charge-separated resonance forms of the C9-keto group to the electronic structure of the cofactor. The presence of a negative charge near BPhL is consistent with the known photochemistry of F(L121)D RCs, which indicates that the free energy of P+BPhL- is substantially higher than in wild-type RCs.

  • the nature and dynamics of the charge separated intermediate in reaction centers in which bacteriochlorophyll replaces the photoactive Bacteriopheophytin 2 the rates and yields of charge separation and recombination
    The Journal of Physical Chemistry, 1995
    Co-Authors: Christine Kirmaier, Craig C Schenck, Laurent Laporte, Dewey Holten
    Abstract:

    The primary photochemistry of the (M)L214H and (M)L214H/(L)E104V mutant bacterial reaction centers (RCs) from Rhodobacter sphaeroides has been investigated at room and cryogenic temperatures. In both mutants the native Bacteriopheophytin electron acceptor (BPh{sub L}) is replaced with a bacteriochlorophyll (BChl) molecule denoted by {Beta}; in the double mutant a hydrogen-bonding interaction of {Beta}-is removed. The initial stage of charge separation, formation of an intermediate P{sup +}I{sup -}, is slowed somewhat in both mutants but without a detectable loss in yield. However, the yield of the subsequent stage of charge separation, P{sup +}I{sup N} {yields} P{sup +}Q{sub A}{sup -}, is significantly reduced due to the combination of slower electron transfer from I{sup -} to Q{sub A} and enhanced charge recombination of P{sup +}I{sup -} to the ground state. Models are considered in which P{sup +}{Beta}{sup -} and P{sup +}BCh1{sub L} are quantum-mechanically mixed or in thermal equilibrium. It is concluded that P{sup +}{Beta}{sup -} and P{sup +}BCh1{sub L} are very close in energy in the mutants and that P{sup +}BCh1{sub L} is very close in energy to the primary electron donor, P{sup *}, in both the mutant and wild-type RCs. 40 refs., 8 figs., 1 tab.

  • charge separation in a reaction center incorporating bacteriochlorophyll for photoactive Bacteriopheophytin
    Science, 1991
    Co-Authors: Christine Kirmaier, Dewey Holten, D Gaul, R Debey, C C Schenck
    Abstract:

    Site-directed mutagenic replacement of M subunit Leu214 by His in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides results in incorporation of a bacteriochlorophyll molecule (BChl) in place of the native Bacteriopheophytin (BPh) electron acceptor. Evidence supporting this conclusion includes the ground-state absorption spectrum of the (M)L214H mutant, pigment and metal analyses, and time-resolved optical experiments. The genetically modified RC supports transmembrane charge separation from the photoexcited BChl dimer to the primary quinone through the new BChl molecule, but with a reduced quantum yield of 60 percent (compared to 100 percent in wild-type RCs). These results have important implications for the mechanism of charge separation in the RC, and rationalize the choice of (bacterio)pheophytins as electron acceptors in a variety of photosynthetic systems.

A J Hoff - One of the best experts on this subject based on the ideXlab platform.

  • the effect of exchange of Bacteriopheophytin a with plant pheophytin a on charge separation in y m210 w mutant reaction centers of rhodobacter sphaeroides at low temperature
    Biochimica et Biophysica Acta, 2003
    Co-Authors: Anatoli Ya Shkuropatov, P Gast, Sieglinde Neerken, Hjalmar P Permentier, Rik De Wijn, Kristiane A Schmidt, V A Shuvalov, Thijs J Aartsma, A J Hoff
    Abstract:

    Abstract The Bacteriopheophytin a molecules at the HA and HB binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of HA−, presumably by raising the free energy level of the P+BA− state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll BA. Exchange of the Bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of HA, inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P+BB− state, where BB is the accessory bacteriochlorophyll in the “inactive” B-branch of the cofactors. The BB molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Qy region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P+BB− state and absorption properties of the BB bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the BB binding pocket.

  • efficient conditions for the photoaccumulation of ha in the photosynthetic reaction centre of rhodobacter sphaeroides r26 with uniformly labelled Bacteriopheophytin monitored by fourier transform infrared difference spectroscopy
    Vibrational Spectroscopy, 1999
    Co-Authors: T A Egorovazachernyuk, P Gast, Andre Remy, Anatoly Ya Shkuropatov, A J Hoff, Klaus Gerwert, Huub J M De Groot
    Abstract:

    Abstract The native Bacteriopheophytins (BPheo) a at sites HA and HB in the reaction centre (RC) of Rhodobacter sphaeroides R26 have been exchanged with uniformly 13 C , 15 N labelled BPheo a. Exchange at the HA site was >50% as monitored with light-induced FTIR-difference spectroscopy indicated by the shift of the band at 1590 cm−1. The photoreduction of HA has been monitored by light-induced FTIR-difference spectroscopy at 22°C in the presence of reductant and mediator: either sodium dithionite and cytochrome c, or sodium dithionite and dyes (potassium indigotetrasulfonate, neutral red). New experimental conditions are described for HA− photoaccumulation, and light-induced FTIR-difference spectra characteristic of HA− are reported. The HA−/HA FTIR-difference spectra of Rb. sphaeroides R26 RCs in which uniformly 13 C , 15 N labelled BPheo a replaces the photoactive BPheo will allow the BPheo modes to be assigned and to be discriminated from those of the RC protein.

  • photosynthetic rcs of rb sphaeroides r26 with uniformly 13 c 15 n labelled Bacteriopheophytin monitored with ftir and solid state mas nmr
    1998
    Co-Authors: Tatiana Egorovazachernyuk, P Gast, Andre Remy, Anatoly Ya Shkuropatov, A J Hoff, Klaus Gerwert, Huub J M De Groot
    Abstract:

    Although the crystal structure of the photosynthetic reaction center (RC) complex has been determined in great detail, with up to 2.65 A resolution [1, 2] several fundamental questions regarding the charge separation mechanism and electron pathway are still unresolved. For example, the precise role of the protein-cofactor interactions in assisting electron transport and prevention of wasteful back reactions is still poorly understood. The RC of Rhodobacter (Rb.) sphaeroides contains two Bacteriopheophytins (BPheo) that are chemically identical but only one, HA, is known to function as an intermediary electron acceptor while the other, HB, appears not to be actively involved in electron transfer [3,4]. Selective isotopic labelling of the cofactors provides a tool for probing the RC structure and the cofactor-protein binding sites using complementary spectroscopic techniques like MAS NMR and FTIR [5-7]. We recently introduced a novel concept of multispin labelling and assignment of 13C CP/MAS NMR resonances of uniformly 13C labelled pheophytin (Pheo) a in Rb. sphaeroides R26 RCs [8]. The present investigation aims at providing a next step into the study of the protein-Bacteriopheophytin (BPheo) interactions in Rb. sphaeroides R26 RCs through the uniform isotope labelling of BPheo (Fig. 1) followed by 1-D 13C CP/MAS NMR and FTIR difference spectroscopy. Photoaccumulation of HA- under reversible conditions required for FTIR studies was reported for the first time by Nabedryk et al. [9], however the procedure for the photoaccumulation of HA appearedto be difficult to handle. Therefore a new method for efficient generating HA- in a film of Rb. sphaeroides R26 RCs was developed [10].

  • reaction centers of rhodobacter sphaeroides r 26 with selective replacement of Bacteriopheophytin a by pheophytin a ii temperature dependence of the quantum yield of p qa and 3p formation
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Eric M Franken, P Gast, A J Hoff, Anatoli Ya Shkuropatov, Sieglinde Neerken, V A Shuvalov, Christof Francke, Thijs J Aartsma
    Abstract:

    Abstract The quantum yield of the formation of the charge-separated state P+QA− in reaction centers (RCs) of Rhodobacter sphaeroides R-26, in which the Bacteriopheophytins in both the active (A) and the inactive (B) branch are replaced by pheophytin (Pheo) a (ΦA,B-exchanged RCs), shows a positive temperature dependence: it is 38±5% between 10 and 60 K, increases with temperature to 72±5% at 200 K and shows a minor additional increase above this temperature. The temperature dependence of the quantum yield of P+QA− formation in ΦA,B-exchanged RCs is modelled in the framework of a reaction scheme with the energy level of P+PheoA− placed above P+BA− (Shkuropatov, A.Ya. and Shuvalov, V.A. (1993) FEBS Lett. 322, 168–172), by the introduction of direct electron transfer from BA− to QA, assisted by a superexchange-mechanism via P+PheoA−. The observed triplet formation of ΦA,B-exchanged RCs with pre-reduced QA at cryogenic temperatures (quantum yield≤12%) is attributed to a residual fraction of RCs in which only ΦB was exchanged for Pheo a. The lack of triplet formation in pre-reduced ΦA,B-exchanged RCs is consistent with our kinetic model, since this predicts that at low temperatures the state P+PheoA− is not populated.

  • reaction centers of rhodobacter sphaeroides r 26 with selective replacement of Bacteriopheophytin by pheophytin a i characterisation of steady state absorbance and circular dichroism and of the p qa state
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Eric M Franken, P Gast, A J Hoff, Anatoli Ya Shkuropatov, Sieglinde Neerken, V A Shuvalov, Christof Francke, Thijs J Aartsma
    Abstract:

    Abstract Bacteriopheophytin (BPheo) a of reaction centers (RCs) of Rhodobacter sphaeroides R-26 has been exchanged with pheophytin (Pheo) a. By varying the incubation temperature of the pigment exchange procedure two types of RCs were obtained, with either only the BPheo in the B-chain (BPheoB) or both BPheoB and BPheoA replaced by Pheo a. For the two RC types absorption and CD spectra at 6 K as well as P+QA− difference spectra at 10 K are compared with those of native RCs. The most pronounced differences are observed in the QY and QX regions of the (B)Pheos. The P+QA− decay halftime is for RCs with Pheo a in both chains 10–15 ms longer than for native RCs and RCs that still have a BPheo in the A-chain, at all temperatures between 10 and 290 K. At low temperatures all three RC types showed biphasic P+QA− recombination.

V A Shuvalov - One of the best experts on this subject based on the ideXlab platform.

  • ftir spectroscopy of the reaction center of chloroflexus aurantiacus photoreduction of the Bacteriopheophytin electron acceptor
    Biochimica et Biophysica Acta, 2011
    Co-Authors: Alexej A Zabelin, V A Shuvalov, V A Shkuropatova, A Shkuropatov
    Abstract:

    Abstract Mid-infrared spectral changes associated with the photoreduction of the Bacteriopheophytin electron acceptor H A in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus ( Cfl. ) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H A − /H A FTIR (1800–1200 cm − 1 ) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter ( Rba. ) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674 cm − 1 in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13 1 -keto carbonyl of H A upon the radical anion H A − formation, exhibits only a very minor upshift to 1675 cm − 1 in Cfl. aurantiacus . In contrast, the absorption band of the 13 1 -keto C = O of H A − is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623 cm − 1 ). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 13 1 -keto C = O of H A with Glu L104 and Gln L143 upon H A − formation and (iii) a possible influence of the protein dielectric environment on the 13 1 -keto C = O stretching frequency of neutral H A . A conformational heterogeneity of the 13 3 -ester C = O group of H A is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.

  • Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of Rhodobacter sphaeroides
    Biochemistry (Moscow), 2010
    Co-Authors: A. G. Yakovlev, V A Shkuropatova, T. A. Shkuropatova, V A Shuvalov
    Abstract:

    Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides . A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B _A ^− and β^−, where β is a bacteriochlorophyll substituting the native Bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the Bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm^−1. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P^+β^− and/or P^+B _A ^− states. The kinetics of Δ A at 1018 nm contains the oscillations at ∼150 cm^−1 and distinct low-frequency oscillations at 20–100 cm^−1; also, the amplitude of the oscillations at 150 cm^−1 is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm^−1 mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The Δ A kinetics at 751 nm reflects the electron transfer to HB with formation of the P^+H _B ^− state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.

  • the effect of exchange of Bacteriopheophytin a with plant pheophytin a on charge separation in y m210 w mutant reaction centers of rhodobacter sphaeroides at low temperature
    Biochimica et Biophysica Acta, 2003
    Co-Authors: Anatoli Ya Shkuropatov, P Gast, Sieglinde Neerken, Hjalmar P Permentier, Rik De Wijn, Kristiane A Schmidt, V A Shuvalov, Thijs J Aartsma, A J Hoff
    Abstract:

    Abstract The Bacteriopheophytin a molecules at the HA and HB binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of HA−, presumably by raising the free energy level of the P+BA− state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll BA. Exchange of the Bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of HA, inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P+BB− state, where BB is the accessory bacteriochlorophyll in the “inactive” B-branch of the cofactors. The BB molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Qy region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P+BB− state and absorption properties of the BB bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the BB binding pocket.

  • reaction centers of rhodobacter sphaeroides r 26 with selective replacement of Bacteriopheophytin a by pheophytin a ii temperature dependence of the quantum yield of p qa and 3p formation
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Eric M Franken, P Gast, A J Hoff, Anatoli Ya Shkuropatov, Sieglinde Neerken, V A Shuvalov, Christof Francke, Thijs J Aartsma
    Abstract:

    Abstract The quantum yield of the formation of the charge-separated state P+QA− in reaction centers (RCs) of Rhodobacter sphaeroides R-26, in which the Bacteriopheophytins in both the active (A) and the inactive (B) branch are replaced by pheophytin (Pheo) a (ΦA,B-exchanged RCs), shows a positive temperature dependence: it is 38±5% between 10 and 60 K, increases with temperature to 72±5% at 200 K and shows a minor additional increase above this temperature. The temperature dependence of the quantum yield of P+QA− formation in ΦA,B-exchanged RCs is modelled in the framework of a reaction scheme with the energy level of P+PheoA− placed above P+BA− (Shkuropatov, A.Ya. and Shuvalov, V.A. (1993) FEBS Lett. 322, 168–172), by the introduction of direct electron transfer from BA− to QA, assisted by a superexchange-mechanism via P+PheoA−. The observed triplet formation of ΦA,B-exchanged RCs with pre-reduced QA at cryogenic temperatures (quantum yield≤12%) is attributed to a residual fraction of RCs in which only ΦB was exchanged for Pheo a. The lack of triplet formation in pre-reduced ΦA,B-exchanged RCs is consistent with our kinetic model, since this predicts that at low temperatures the state P+PheoA− is not populated.

  • reaction centers of rhodobacter sphaeroides r 26 with selective replacement of Bacteriopheophytin by pheophytin a i characterisation of steady state absorbance and circular dichroism and of the p qa state
    Biochimica et Biophysica Acta, 1997
    Co-Authors: Eric M Franken, P Gast, A J Hoff, Anatoli Ya Shkuropatov, Sieglinde Neerken, V A Shuvalov, Christof Francke, Thijs J Aartsma
    Abstract:

    Abstract Bacteriopheophytin (BPheo) a of reaction centers (RCs) of Rhodobacter sphaeroides R-26 has been exchanged with pheophytin (Pheo) a. By varying the incubation temperature of the pigment exchange procedure two types of RCs were obtained, with either only the BPheo in the B-chain (BPheoB) or both BPheoB and BPheoA replaced by Pheo a. For the two RC types absorption and CD spectra at 6 K as well as P+QA− difference spectra at 10 K are compared with those of native RCs. The most pronounced differences are observed in the QY and QX regions of the (B)Pheos. The P+QA− decay halftime is for RCs with Pheo a in both chains 10–15 ms longer than for native RCs and RCs that still have a BPheo in the A-chain, at all temperatures between 10 and 290 K. At low temperatures all three RC types showed biphasic P+QA− recombination.

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  • Bacteriopheophytin triplet state in rhodobacter sphaeroides reaction centers
    Photosynthesis Research, 2016
    Co-Authors: Rafal Bialek, Gotard Burdzinski, Michael R Jones, Krzysztof Gibasiewicz
    Abstract:

    It is well established that photoexcitation of Rhodobacter sphaeroides reaction centers (RC) with reduced quinone acceptors results in the formation of a triplet state localized on the primary electron donor P with a significant yield. The energy of this long-lived and therefore potentially damaging excited state is then efficiently quenched by energy transfer to the RC spheroidenone carotenoid, with its subsequent decay to the ground state by intersystem crossing. In this contribution, we present a detailed transient absorption study of triplet states in a set of mutated RCs characterized by different efficiencies of triplet formation that correlate with lifetimes of the initial charge-separated state P+H A − . On a microsecond time scale, two types of triplet state were detected: in addition to the well-known spheroidenone triplet state with a lifetime of ~4 μs, in some RCs we discovered a Bacteriopheophytin triplet state with a lifetime of ~40 μs. As expected, the yield of the carotenoid triplet increased approximately linearly with the lifetime of P+H A − , reaching the value of 42 % for one of the mutants. However, surprisingly, the yield of the Bacteriopheophytin triplet was the highest in RCs with the shortest P+H A − lifetime and the smallest yield of carotenoid triplet. For these the estimated yield of Bacteriopheophytin triplet was comparable with the yield of the carotenoid triplet, reaching a value of ~7 %. Possible mechanisms of formation of the Bacteriopheophytin triplet state are discussed.

  • early Bacteriopheophytin reduction in charge separation in reaction centers of rhodobacter sphaeroides
    Biophysical Journal, 2013
    Co-Authors: Ivo H M Van Stokkum, Michael R Jones, Laura Paparelli, Marie Louise Groot
    Abstract:

    A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and Bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA− band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between BA and HA, and the effect of protein conformation on the electron transfer rate.

  • replacement or exclusion of the b branch Bacteriopheophytin in the purple bacterial reaction centre the h b cofactor is not required for assembly or core function of the rhodobacter sphaeroides complex
    Biochimica et Biophysica Acta, 2005
    Co-Authors: Ashley J Watson, Paul K Fyfe, Dmitrij Frolov, Marion C Wakeham, Eliane Nabedryk, Rienk Van Grondelle, Jacques Breton, Michael R Jones
    Abstract:

    All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the Bacteriopheophytin (HB) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the HB Bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the HB position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the Abranch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an HB bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the HB cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (QB) site. The data are discussed with respect to possible functional roles of the HB Bacteriopheophytin, and we conclude that the reason(s) for conservation of a Bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.

  • Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides
    Photosynthesis Research, 1999
    Co-Authors: Justin P. Ridge, Marion E. Van Brederode, Matthew G. Goodwin, Rienk Van Grondelle, Michael R Jones
    Abstract:

    Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor Bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor Bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.

  • reorientation of the acetyl group of the photoactive Bacteriopheophytin in reaction centers of rhodobacter sphaeroides an endor triple resonance study
    Biospectroscopy, 1999
    Co-Authors: Michael R Jones, Wolfgang Lubitz
    Abstract:

    The freeze-trapped Bacteriopheophytin a radical anion Φ has been investigated by 1H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of Φ, denoted I and I, can be stabilized below 200 K. The state I is metastable and relaxes to I as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of Φ between the two states is interpreted in terms of a conformational change of ΦA after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the Bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one Φ state is obtained that is distinct from I and I, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 31-keto oxygen of ΦA that impedes the reorientation of the acetyl group. Only one Φ state is observed in the YL(M210) reaction center, which is similar to the metastable state I in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 35–46, 1999