Primary Electron Acceptor Research Articles

Characterization of the Secondary Quinone (QB) Binding Pocket in Photosynthetic Reaction Centers Using Pulsed EPR Spectroscopy

3-pulse ESEEM and HYSCORE pulse sequences have been used to analyze the secondary electron acceptor semiquinone anion radical (QB-). Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone molecules functioning as primary and secondary electron acceptors. The primary quinone radical (QA-) has been extensively studied, and hydrogen bonds have been characterized at both carbonyls. The structure around QB- has received less attention. The O4 carbonyl has been suggested to be hydrogen bonded to the Nδ from a histidine at residue L190. The O1 carbonyl also possesses a hydrogen bond that is weaker than that at O4. OH from serine at L223 is important in the hydrogen bond structure at this carbonyl. However, contributions from surrounding peptide nitrogens are suggested by x-ray structures but have not yet been investigated by EPR methods. Pulsed EPR studies of the QB- radical confirm one strongly coupled nitrogen with NQI frequencies consistent with a histidine Nδ. 3-pulse ESEEM and HYSCORE spectra also contain peaks from a second nitrogen nucleus. A priori knowledge of the origins of these peaks is less clear, but could include contributions from a backbone nitrogen. Additionally, NQI modulation from 14N is sufficiently shallow to observe signals from 2 protons in HYSCORE measurements. These results suggest the possibility that an additional residue contributes to the stability of QB-. Supported by NSF grant MCB 08-18121.

2P289 1A1325 種々の生物における光化学系II電子受容体キノンの酸化還元電位の分光電気化学計測(光生物-光合成,第48回日本生物物理学会年会)

2P289 1A1325 種々の生物における光化学系II電子受容体キノンの酸化還元電位の分光電気化学計測(光生物-光合成,第48回日本生物物理学会年会)

Hydrogen Bond Interactions of the Pheophytin Electron Acceptor and Its Radical Anion in Photosystem II As Revealed by Fourier Transform Infrared Difference Spectroscopy

The primary electron acceptor pheophytin (Pheo(D1)) plays a crucial role in regulation of forward and backward electron transfer in photosystem II (PSII). It is known that some cyanobacteria control the Pheo(D1) potential in high-light acclimation by exchanging the D1 proteins from different copies of the psbA genes. To clarify the mechanism of the potential control of Pheo(D1), we studied the hydrogen bond interactions of Pheo(D1) in the neutral and anionic states using light-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra of Pheo(D1) upon its photoreduction were obtained using three different PSII preparations, PSII core complexes from Thermosynechococcus elongatus possessing PsbA1 as a D1 subunit (PSII-PsbA1), those with PsbA3 (PSII-PsbA3), and PSII membranes from spinach. The D1-Gln130 side chain, which is hydrogen bonded to the 13(1)-keto C=O group of Pheo(D1) in PSII-PsbA1, is replaced by Glu in PSII-PsbA3 and spinach PSII. The spectrum of PSII-PsbA1 exhibited 13(1)-keto C=O bands at 1682 and 1605 cm(-1) in neutral Pheo(D1) and its anion, respectively, while the corresponding bands were observed at frequencies lower by 1-3 and 18-19 cm(-1), respectively, in the latter two preparations. This larger frequency shift in Pheo(D1)(-) than Pheo(D1) by the change of the hydrogen bond donor was well reproduced by density functional theory (DFT) calculations for the Pheo models hydrogen bonded with acetamide and acetic acid. The DFT calculations also exhibited a higher redox potential for Pheo reduction in the model with acetic acid than that with acetamide, consistent with previous observations for the D1-Gln130Glu mutant of Synechocystis. It is thus concluded that a stronger hydrogen bond effect on the Pheo(-) anion than the neutral Pheo causes the shift in the redox potential, which is utilized in the photoprotection mechanism of PSII.

Application of the standard theory of electronic transitions to the description of oscillations in the kinetics of electron transfer in reaction centers of purple bacteria

The standard theory of the electron transfer between donor and acceptor molecules was used to describe oscillations in the reduction kinetics of the intermediate electron acceptor BA and the primary electron acceptor HA. The kinetics of the reduction of BA and HA were simulated on the basis of the model in which one and two accepting modes were used. A principal experiment is offered for the selection of the suitable theory for adequate description of oscillations in the kinetics of electron transfer in the reaction centers of purple bacteria.

Redox Potential of the Primary Plastoquinone Electron Acceptor QA in Photosystem II from Thermosynechococcus elongatus Determined by Spectroelectrochemistry

The redox potential of the primary plastoquinone electron acceptor Q(A), E(m)(Q(A)/Q(A)(-)), in an oxygen-evolving photosystem (PS) II complex from a thermophilic cyanobacterium Thermosynechococcus elongatus was determined to be -140 +/- 2 mV vs. SHE by thin-layer cell spectroelectrochemistry for the first time. The E(m)(Q(A)/Q(A)(-)) value obtained here together with the recently determined redox potential of pheophytin (Phe) a [Kato et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 17365-17370] yields -330 to -370 mV for the free energy change by electron transfer from Phe a(-) to Q(A) and provides a renewed picture for the energetics on the electron acceptor side in PS II.

Spectroelectrochemical determination of the redox potential of pheophytin a, the primary electron acceptor in photosystem II.

Thin-layer cell spectroelectrochemistry, featuring rigorous potential control and rapid redox equilibration within the cell, was used to measure the redox potential E(m)(Phe a/Phe a(-)) of pheophytin (Phe) a, the primary electron acceptor in an oxygen-evolving photosystem (PS) II core complex from a thermophilic cyanobacterium Thermosynechococcus elongatus. Interferences from dissolved O(2) and water reductions were minimized by airtight sealing of the sample cell added with dithionite and mercury plating on the gold minigrid working electrode surface, respectively. The result obtained at a physiological pH of 6.5 was E(m)(Phe a/Phe a(-)) = -505 + or - 6 mV vs. SHE, which is by approximately 100 mV more positive than the values measured approximately 30 years ago at nonphysiological pH and widely accepted thereafter in the field of photosynthesis research. Using the P680* - Phe a free energy difference, as estimated from kinetic analyses by previous authors, the present result would locate the E(m)(P680/P680(+)) value, which is one of the key parameters but still resists direct measurements, at approximately +1,210 mV. In view of these pieces of information, a renewed diagram is proposed for the energetics in PS II.

Effect of the P700 pre-oxidation and point mutations near A 0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700 +). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A 0, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700 +. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼ 7 ps and ∼ 25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A 0: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A 0. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700 +. The same charge is proposed to be responsible for the fast A +A 0 − → AA 0 charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A 0 axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A +A 0 −.

Higher concentration of QB-nonreducing photosystem II centers in triazine-resistant Chenopodium album plants as revealed by analysis of chlorophyll fluorescence kinetics

Plants resistant to triazine-type herbicides are known to be altered in their photosystem II reaction center. Serine at site 264 in D1 protein is replaced by glycine. The measurements of chlorophyll a fluorescence excitations with a variable number of saturating flashes in Chenopodium album plants show characteristic differences between the resistant and the wild-type plants. These differences appear in response to the first flash as well as in the rise pattern of subsequent flashes of a 12.5 Hz flash train. The differences indicate a higher concentration of Q(B)-nonreducing reaction centers in the resistant biotype, and confirm earlier results on a slower rate of electron transport between the primary and secondary electron acceptors.

Evidence of specialized bromate-reducing bacteria in a hollow fiber membrane biofilm reactor

Bromate is a carcinogenic disinfection by-product formed from bromide during ozonation or advanced oxidation. We previously observed bromate reduction in a hydrogen-based, denitrifying hollow fiber membrane biofilm reactor (MBfR). In this research, we investigated the potential existence of specialized bromate-reducing bacteria. Using denaturing gradient gel electrophoresis (DGGE), we compared the microbial ecology of two denitrifying MBfRs, one amended with nitrate as the electron acceptor and the other with nitrate plus bromate. The DGGE results showed that bromate exerted a selective pressure for a putative, specialized bromate-reducing bacterium, which developed a strong presence only in the reactor with bromate. To gain further insight into the capabilities of specialized, bromate-reducing bacteria, we explored bromate reduction in a control MBfR without any primary electron acceptors. A grown biofilm in the control MBfR reduced bromate without previous exposure, but the rate of reduction decreased over time, especially after perturbations resulting in biomass loss. The decrease in bromate reduction may have been the result of the toxic effects of bromate. We also used batch tests of the perchlorate-reducing pure culture, Dechloromonas sp. PC1 to test bromate reduction and growth. Bromate was reduced without measurable growth. Based on these results, we speculate bromate's selective pressure for the putative, specialized BRB observed in the DGGE was not growth related, but possibly based on resistance to bromate toxicity.

Type I reaction center from the green sulfur bacterium Chlorobium tepidum: is Chl a a primary electron acceptor?

The green sulfur bacterium Chlorobium tepidum has one of the simplest type I reaction center (RC) complexes. While its structure is still unknown, biochemical and protein sequence analyses suggest that it is similar to photosystem I (PS I), with two BChl a forming a special pair P840, four Chl a serving as pairs of accessory and primary electron acceptor (A0) pigments and 14 BChl a constituting as an immediate RC antenna. This is a dramatic simplification compared to PS I RC, where 90 Chl a antenna pigments serve as antenna and 6 additional Chl a molecules function as electron transfer cofactors. The resulting spectral congestion has prevented direct visualization of ultrafast electron transfer processes within PS I RC and even the sequence of primary electron transfer processes in PS I is still under debate. The suggested presence of two types of pigments in RC from Chlorobium tepidum removes spectral congestion and opens a way to directly visualize electron transfer steps in type I RC using ultrafast spectroscopy, since the Chl a and BChl a pigments absorb at ∼670 nm and ∼800 nm, respectively. To confirm the proposed functional role of Chl a as electron transfer cofactor we performed extensive ultrafast optical pump-probe experiments on different preparations of RC complexes from Chlorobium tepidum, revealing energy/electron transfer rates between different groups of pigments. Surprisingly, we found that ∼3 out of 4 Chl a pigments do not transfer excitation energy to the BChl a antenna or to P840, which indicates that these pigments must be >20A away from any other BChl a pigment and thus argues against the suggested presence of 4 Chl a in the reaction center core complex.

Kinetics and Energetics of Electron Transfer Reactions in a Photosynthetic Bacterial Reaction Center Assembled with Zinc Bacteriochlorophylls

Electron transfer processes were studied in the reaction center (RC) of a Rhodobacter sphaeroides magnesium chelatase (bchD) mutant that assembles with six chemically identical chlorin molecules. A previous study [Jaschke & Beatty, Biochemistry, 2007] and this work show the complete absence of bacteriochlorophyll (containing Mg as the metal) and bacteriopheophytin from the bchD mutant RC. Instead, bacteriochlorophylls containing a Zn atom as the metal (Zn-BChl) occupy the binding sites of the special pair (P), accessory bacteriochlorophyll (B), and primary electron acceptor (H). In spite of significant differences in cofactor composition, electron transfer from excited P through B to H proceeds with high efficiency and with rates nearly identical to the wild type RC. The rate of electron transfer from H to QA is also the same as that observed in the wild type RC. Thus, the protein-cofactor interactions, mainly through electron sharing between the metal of the BChl and the protein, play an important role in adjusting the energies of the cofactors to form an efficient electron transfer system. The study also suggests that the overall electron transfer from P to H is more sensitive to the energy change between P and B than B and H, and can tolerate a large variation in the redox energy of H.

Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation

Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A(max) 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH<7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using (18)O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.

1P-232 QA再構成とFTIR解析によるPSIIにおける第一電子受容体QAの分子間相互作用の解明(光生物-光合成,第47回日本生物物理学会年会)

1P-232 QA再構成とFTIR解析によるPSIIにおける第一電子受容体QAの分子間相互作用の解明(光生物-光合成,第47回日本生物物理学会年会)

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H(A)(-). The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P(A) and P(B) in the P(+) state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P(A) and P(B) macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P(+)Q(-)/PQ spectra suggests the 17(3)-ester group of the bacteriochlorophyll P(A) to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B(A) and the bacteriopheophytin H(A). Structural changes are observed in the monomer bacteriochlorophyll B(B) binding site in the inactive chromophore branch of the mutant RCs.

Electron transfer from A−0 to A1 in Photosystem I from Chlamydomonas reinhardtii occurs in both the A and B branch with 25–30-ps lifetime

We have recorded transient absorption kinetics at 390 nm with picosecond resolution in order to observe electron transfer from the reduced primary acceptor, A, to the secondary acceptor, A(1), in wild type and mutated Photosystem I from Chlamydomonas reinhardtii. In the mutants, the methionine axial ligand to the primary electron acceptor in either the A- or B-branch of electron transfer cofactors, was replaced with histidine. Both of the mutations reduced the formation of a positive signal at 390 nm, characteristic of A to a level approximately half of that observed in wild type Photosystem I. It is concluded that in the mutated branch of Photosystem I, electron transfer from A to A(1) does not occur. The absorption kinetics resulting from subtraction of either of the mutants' traces from that of wild type is interpreted to reflect the kinetics of A- or B-side electron transfer from A to A(1) in the the wild type Photosystem I. Each of these traces could be fitted with a monoexpoenential decay characterized by the same amplitude and 25-30-ps lifetime. The almost identical effect of both mutations on A formation confirm a similar engagement of both the A- ad B-branches in electron transfer to A(1) in Photosystem I from C. reinhardtii. This observation is in contrast to the unidirectional electron transfer concluded from the studies on similar mutants of cyanobacterial Photosystem I.(1) Thus, this contribution provides further evidence for functional differences between these two model Photosystems.

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5–4.8 times depending on the used electron acceptor. The apparent K m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD + on activity showed that the E95Q mutation caused a 2.4-fold decrease of K i NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD + by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K i ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD +. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from − 350 mV to − 310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.

Heterologous Production, Isolation, Characterization and Crystallization of a Soluble Fragment of the NADH:Ubiquinone Oxidoreductase (Complex I) from Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chains in many bacteria and most eukaryotes. It is the least understood of all, due to its enormous size and unique energy conversion mechanism. The bacterial complex is in general made up of 14 different subunits named NuoA-N. Subunits NuoE, -F, and -G comprise the electron input part of the complex. We have cloned these genes from the hyperthermophilic bacterium Aquifex aeolicus and expressed them heterologously in Escherichia coli. A soluble subcomplex made up of NuoE and NuoF and containing the NADH binding site, the primary electron acceptor flavin mononucleotide (FMN), the binuclear iron-sulfur cluster N1a, and the tetranuclear iron-sulfur cluster N3 was isolated by chromatographic methods. The proteins were identified by N-terminal sequencing and mass spectrometry; the cofactors were characterized by UV/vis and EPR spectroscopy. Subunit NuoG was not produced in this strain. The preparation was thermostable and exhibited maximum NADH/ferricyanide oxidoreductase activity at 85 degrees C. Analytical size-exclusion chromatography and dynamic light scattering revealed the homogeneity of the preparation. First attempts to crystallize the preparation led to crystals diffracting more than 2 A.

Removal of the PsaF Polypeptide Biases Electron Transfer in Favor of the PsaB Branch of Cofactors in Triton X‐100 Photosystem I Complexes from Synechococcus sp. PCC 7002†

Continuous wave (CW) and transient electron paramagnetic resonance studies have implied that when PsaF is removed genetically, the double reduction of A1A is facile, the lifetime of A1A(-) is shorter and the ratio of fast to slow kinetic phases increases in PS I complexes isolated with Triton X-100 (Van der Est, A., A. I. Valieva, Y. E. Kandrashkin, G. Shen, D. A. Bryant and J. H. Golbeck [2004] Biochemistry43, 1264-1275). Changes in the lifetimes of A1A(-) and A1B(-) are characteristic of mutants involving the quinone binding sites, but changes in the relative amplitudes of A1A(-) and A1B(-) are characteristic of mutants involving the primary electron acceptors, A0A and A0B. Here, we measured the fast and slow phases of electron transfer from A1B(-) and A1A(-) to FX in psaF and psaE psaF null mutants using time-resolved CW and pump-probe optical absorption spectroscopy. The lifetime of the fast kinetic phase was found to be unaltered, but the lifetime of the slow kinetic phase was shorter in the psaF null mutant and even more so in the psaE psaF null mutant. Concomitantly, the amplitude of the fast kinetic phase increased by a factor of 1.8 and 2.0 in the psaF and psaE psaF null mutants, respectively, at the expense of the slow kinetic phase. The change in ratio of the fast to slow kinetic phases is explained as either a redirection of electron transfer through A1B at the expense of A1A, or a shortening of the lifetime of A1A(-) to become identical to that of A1B(-). The constant lifetime and the characteristics of the near-UV spectrum of the fast kinetic phase favor the former explanation. A unified hypothesis is presented of a displacement of the A-jk1 alpha-helix and switchback loop, which would weaken the H-bond from Leu722 to A1A, accounting for the acceleration of the slow kinetic phase, as well as weaken the H-bond from Tyr696 to A0A, accounting for the bias of electron transfer in favor of the PsaB branch of cofactors.

Unique photosystems in Acaryochloris marina

A short overview is given on the discovery of the chlorophyll d-dominated cyanobacterium Acaryochloris marina and the minor pigments that function as key components therein. In photosystem I, chlorophyll d', chlorophyll a, and phylloquinone function as the primary electron donor, the primary electron acceptor and the secondary electron acceptor, respectively. In photosystem II, pheophytin a serves as the primary electron acceptor. The oxidation potential of chlorophyll d was higher than that of chlorophyll a in vitro, while the oxidation potential of P740 was almost the same as that of P700. These results help us to broaden our view on the questions about the unique photosystems in Acaryochloris marina.

Evidence of specialized bromate-reducing bacteria in a hollow fiber membrane biofilm reactor

Bromate is a carcinogenic disinfection by-product formed from bromide during ozonation or advanced oxidation of drinking water. We previously observed bromate reduction in a hydrogen-based, denitrifying hollow fiber membrane biofilm reactor (MBfR). In this research, we investigated the potential existence of specialized bromate-reducing bacteria. Using denaturing gradient gel electrophoresis (DGGE), we compared the microbial ecology of two denitrifying MBfRs, one amended with nitrate as the electron acceptor and the other with nitrate plus bromate. The DGGE results showed that bromate exerted a selective pressure for a putative, specialized bromate-reducing bacterium, which developed a strong presence only in the reactor with bromate. To gain further insight into the capabilities of specialized, bromate-reducing bacteria, we explored bromate reduction in a control MBfR without any primary electron acceptors. A grown biofilm in the control MBfR reduced bromate without previous exposure, but the rate of reduction decreased over time, especially after perturbations resulting in biomass loss. The decrease in bromate reduction may have been the result of the toxic effects of bromate. We also used batch tests of the perchlorate-reducing pure culture, Dechloromonas sp. PC1 to test bromate reduction and growth. Bromate was reduced without measurable growth. Based on these results, we speculate bromate's selective pressure for the putative, specialized BRB observed in the DGGE was not growth related, but possibly based on resistance to bromate toxicity.