Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13¹-deoxo-13¹-hydroxy-pheophytin a (13¹-OH-Pheo). The absorption bands of 13¹-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC_1×) or twice (RC_2×). HPLC analysis and Pheo Q_X absorption at 543 nm show that in RC_1× about 50% of Pheo is replaced and in RC_2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo⁻ is unmodified in RC_1× and decreased only in RC_2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q_Y region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC_1× or RC_2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mg‐dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY‐GREEN (SGR) gene encodes Mg‐dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion‐mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F_v/F_m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light‐harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.     

Role of the electrostatic energy between the photo-products and ionic groups on the photoinduced electron transfer rates from aromatic amino acids to the excited flavin in five single-point substitution isoforms of the charged amino acid residue-13 in the FMN-binding protein

Effect of the charge (negative, positive or neutral) of amino acid residue-13 on the photoinduced electron transfer (ET) from Trp32, Tyr35 and Trp106 to the excited isoalloxazine was evaluated in the flavin mononucleotide-binding protein from Desulfovibrio vulgaris isolate Miyazaki F (DvFBP). The protein structures of the wild type and the four isoforms where glutamic acid-13 is replaced with lysine (E13K), arginine (E13R), threonine (E13T) and glutamine (E13Q) in aqueous solution were obtained by molecular dynamics simulation. The distances between the amino acid residue-13 and isoalloxazine (Iso), and between the amino acid residue-13 and the ET donors were longer than 1 nm. The ET rates were evaluated with the Kakitani and Mataga model (KM theory) from their ultrafast fluorescence dynamics by means of a non-linear least squares method. Electrostatic (ES) energies between the photo-products and other ionic groups in the proteins markedly varied among ET donors and among the DvFBP isoforms, while the other physical quantities related to the ET rates, the solvent reorganisation and ES energies between the Iso anion and the donor cations did not vary much between the proteins and donors. A plot of the logarithmic ET rates versus either the total free energy gaps or the net ES energies between the photo-products and the other ionic groups both displayed a parabolic function and so the net ES energies are an important influential factor upon the ET rate, in addition to the donor–acceptor distance.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Cytochrome oxidation in bacterial photosynthesis

In this paper we propose that the reduction of the bacteriochlorophyl dimer cation (P⁺) by cytochrome c in the photosynthetic bacteria Rps. viridis and Chromatium vinosum proceeds via two parallel electron transfer (ET) processes from two distinct cytochrome c molecules. The dominating ET process at high temperatures involves the activated oxidation of the high-potential cytochrome c at closest proximity to P, while the dominating low-temperature process involves activationless ET from a low-potential cytochrome c, which is further away from P. The available data for the effects of blocking the low-potential cytochrome c on ET dynamics are consistent with this model, which results in reasonable nuclear reorganization and electronic coupling parameters for the parallel cytochrome oxidation processes. The lack of universality in the cytochrome oxidation in reaction centres of various bacteria is emphasized.     

Pigment exchange of Photosystem II reaction center by chlorophyll d

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

Kinetics and efficiency of excitation energy transfer from chlorophylls,their heavy metal-substituted derivatives,and pheophytins to singlet oxygen

Time-resolved measurements of the singlet oxygen infrared (1269 nm) luminescence were used to follow the kinetics and efficiency of excitation energy transfer (EET) between chlorophyll (Chl) derivatives and oxygen in acetone. The studied pigments were Mg-Chl a and b and their heavy metal (Cu²⁺ and Zn²⁺)-substituted analogues, as well as pheophytin (Pheo) a and b. The efficiency of EET from chlorophyll to oxygen was highly dependent on the central ion in the pigment. Cu-Chl a and Cu-Chl b had the lowest efficiencies of singlet oxygen production, while Pheo a had a higher one, and Zn-Chl a had a similar one compared to Mg-Chl a. Also the side chain (position C-7, i.e. Chl a vs. Chl b) influenced the efficiency of singlet oxygen formation. In the case of square-planar complexes like Cu-Chl and Pheo, EET was more efficient in the Chl a derivatives than in those of Chl b; the opposite effect was observed in the case of the five- or six-coordinated Mg-Chl and Zn-Chl. As for the lifetime of the Chl triplet state, the most striking difference to Mg-Chl again was found in the case of Cu-Chls, which had much shorter lifetimes. Furthermore, the central ion in Chl affected the physical quenching of singlet oxygen: its efficiency was decreasing from Mg-Chl through Zn-Chl over Cu-Chl to Pheo. The results are discussed in the context of the oxidative stress accompanying heavy metal-induced stress in plants.     

Modulating the redox potential of the stable electron acceptor, Q(B), in mutagenized photosystem II reaction centers

One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 ? cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.     

Characteristics of mixed association and deactivation of electron excitation in chlorophyll-pheophytin complexes

The regularities of the individual and mixed association of chlorophylls (Chl a, PChl a) with pheophytin (Pheo) were investigated. The complex studies of optical activity, spectral--luminescent and energetic characteristics of aggregates were carried out in mixture of solvents aceton-water (1:49). The formation of pigment mixed associates leads to intracomplex energy transfer from Chl (or PChl) to Pheo. It is shown that the efficiencies of such process, determined by independent ways via the luminescence quenching of energy donor or the emission sensibilization of acceptor, are identical. The energy migration mechanism is the inductive resonance one in studied complexes. The main patterns of the electronic excitation energy deactivation in such systems are discussed. The obtained results are analysed taking into account the contemporary background of the role of pheophytin in the primary processes of photosynthesis.     

Insight into the redox partner interaction mechanism in cytochrome P450BM‐3 using molecular dynamics simulations

Flavocytochrome P450BM‐3 is a soluble bacterial reductase composed of two flavin (FAD/FMN) and one HEME domains. In this article, we have performed molecular dynamics simulations on both the isolated FMN and HEME domains and their crystallographic complex, with the aim to study their binding modes and to garner insight into the interdomain electron transfer (ET) mechanism. The results evidenced an interdomain conformational rearrangement that reduces the average distance between the FMN and HEME cofactors from 1.81 nm, in the crystal structure, to an average value of 1.41 ± 0.09 nm along the simulation. This modification is in agreement with previously proposed hypotheses suggesting that the crystallographic FMN/HEME complex is not in the optimal arrangement for favorable ET rate under physiological conditions. The calculation of the transfer rate along the simulation, using the Pathways Path method, demonstrated the occurrence of seven ET pathways between the two redox centers, with three of them providing ET rates (K_ET) comparable with the experimental one. The sampled ET pathways comprise the amino acids N319, L322, F390, K391, P392, F393, A399, C400, and Q403 of the HEME domain and M490 of the FMN domain. The values of K_ET closer to the experiment were found along the pathways FMN(C7) → F390 → K391 → P392 → HEME(Fe) and FMN(C8) → M490 → F393 → HEME(Fe). Finally, the analysis of the collective modes of the protein complex evidences a clear correlation of the first two essential modes with the activation of the most effective ET pathways along the trajectory. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 197–209, 2014.     

Protein–cofactor binding and ultrafast electron transfer in riboflavin binding protein under the spatial confinement of nanoscopic reverse micelles

In this contribution, we study the effect of confinement on the ultrafast electron transfer (ET) dynamics of riboflavin binding protein (RBP) to the bound cofactor riboflavin (Rf, vitamin B2), an important metabolic process, in anionic sodium bis(2‐ethylhexyl) sulfosuccinate reverse micelles (AOT‐RMs) of various hydration levels. Notably, in addition to excluded volume effect, various nonspecific interactions like ionic charge of the confining surface can influence the biochemical reactions in the confined environment of the cell. To this end, we have also studied the ET dynamics of RBP–Rf complex under the confinement of a cationic hexadecyltrimethylammonium bromide (CTAB) RMs with similar water pool size to the anionic AOT‐RMs towards simulating equal restricted volume effect. It has been found that the spatial confinement of RBP in the AOT‐RM of w₀ = 10 leads to the loss of its tertiary structure and hence vitamin binding capacity. Although, RBP regains its binding capacity and tertiary structure in AOT‐RMs of w₀ ≥20 due to its complete hydration, the ultrafast ET from RBP to Rf merely occurs in such systems. However, to our surprise, the ET process is found to occur in cationic CTAB‐RMs of similar volume restriction. It is found that under the spatial confinement of anionic AOT‐RM, the isoalloxazine ring of Rf is improperly placed in the protein nanospace so that ET between RBP and Rf is not permitted. This anomaly in the binding behaviour of Rf to RBP in AOT‐RMs is believed to be the influence of repulsive potential of the anionic AOT‐RM surface to the protein. Our finding thus suggests that under similar size restriction, both the hydration and surface charge of the confining volume could have major implication in the intraprotein ET dynamics in real cellular environments. Copyright © 2013 John Wiley & Sons, Ltd.     

Excitation energy transfer and charge separation in the isolated Photosystem II reaction center

The nature of excitation energy transfer and charge separation in isolated Photosystem II reaction centers is an area of considerable interest and controversy. Excitation energy transfer from accessory chlorophyll a to the primary electron donor P680 takes place in tens of picoseconds, although there is some evidence that thermal equilibration of the excitation between P680 and a subset of the accessory chlorophyll a occurs on a 100-fs timescale. The intrinsic rate for charge separation at low temperature is accepted to be ca. (2 ps)^–1, and is based on several measurements using different experimental techniques. This rate is in good agreement with estimates based on larger sized particles, and is similar to the rate observed with bacterial reaction centers. However, near room temperature there is considerable disagreement as to the observed rate for charge separation, with several experiments pointing to a ca. (3 ps)^–1 rate, and others to a ca. (20 ps)^-1 rate. These processes and the experiments used to measure them will be reviewed.Abbreviations Chl chlorophyll - FWHM full-width at half-maximum - Pheo pheophytin - PS II Photosystem II - P680 primary electron donor of the Photosystem II reaction center - RC reaction center The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

Theoretical studies on the mechanism of primary electron transfer in the photosynthetic reaction center of Rhodobacter sphaeroides

The mechanism of the primary electron transfer (ET) process in the photosynthetic reaction center (PRC) of Rhodobacter sphaeroides has been studied with quantum chemistry method of ab initio density functional theory (DFT) (B3LYP/6-31G) based on the optimized X-ray crystallographic structure. The calculation was carried out on different structural levels. The electronic structure of pigment molecules was first studied, and then the influence of the neighboring protein was taken into account at three approximation levels: (a) the surrounding proteins were treated as a homogeneous medium with a uniform dielectric constant (SCRF); (b) both the influence of axial coordination of His to the special pair P and ABChl as, and the hydrogen bonds between related residues and P and also BPhas were included; and (c) the influence of the electronic structure of the protein subunit chains as a whole was studied. The results suggest that: (1) according to the composition of the HOMO and LUMO of P, there might be a charge-separated state of (BChl_L ⁺BChl_M ⁻) for the excited state of P; (2) to treat the protein surroundings as a homogeneous medium is not sufficient. Different interactions between pigment molecules and related residues play different roles in the ET process; (3) the axial coordination of His to P raises the E _LUMO of P greatly, and it is very important for the ET process to occur in the PRC of wild-type bacterium; the axial coordination of His to ABChl as also raises their E _LUMO significantly; (4) the hydrogen-bonds between amino acid residues and P and also BPh as depress the E _LUMO of the pigment molecules to some extent, which makes the E _LUMO of P lower than those of ABChlas, and the E _LUMO of BPh a _L lower than that of BPh a _M. Consequently, the ET process from P to BPh a _L does not, according to our calculation model, occur via ABChl a _L. The possibility of the ET pathway from P to BPh a _L via ABChl a _L was discussed; (5) the frontier orbitals of protein subunit chains L and M are localized at the random coil area and the α–helix areas, respectively. Results mentioned above support the fact that the ET process proceeds in favourable circumstances along the branch L. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

Evaluation of the selective and differential ET medium for detection of Edwardsiella tarda in aquaculture systems

Aims: Edwardsiella tarda is an important pathogen in aquaculture where it can cause serious losses. A rapid detection of it is vital to minimize the mortalities caused by this disease, and in this work, the effectiveness of the selective differential Edw. tarda medium (ET) was evaluated for the diagnosis of edwardsiellosis as well as for its possible use in epidemiological studies. Methods and Results: ET medium was evaluated in parallel with the commercial Salmonella–Shigella agar (SS), which is usually employed for the selective isolation of enteric bacilli. Moreover, two general media (TSA‐1 and MA) were employed as a control. The results obtained showed that ET is distinctly selective for the isolation of Edw. tarda, allowing its recovery from mixed cultures and natural samples as a unique species. In contrast, although colonies of Edw. tarda could be clearly distinguishable in SS because of the appearance of a characteristic black centre, other enteric and nonenteric bacterial species were also able to grow on this medium. Conclusions: We recommend ET agar as an useful medium for the primary isolation of Edw. tarda from aquaculture samples. Significance and Impact of the Study: The results obtained support ET medium as the most appropriate to develop epidemiological studies of edwardsiellosis in aquaculture and permits an earlier diagnosis of this important disease.     

Rapid and simple isolation of pure photosystem II core and reaction center particles from spinach

Pure and active oxygen-evolving PS II core particles containing 35 Chl per reaction center were isolated with 75% yield from spinach PS II membrane fragments by incubation with n-dodecyl--D-maltoside and a rapid one step anion-exchange separation. By Triton X-100 treatment on the column these particles could be converted with 55% yield to pure and active PS II reaction center particles, which contained 6 Chl per reaction center.Abbreviations Bis-Tris bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane - Chl chlorophyll - CP29 Chl a/b protein of 29 kDa - Cyt b ₅₅₉ cytochrome b ₅₅₉ - DCBQ 2,5-dichloro-p-benzo-quinone - LHC II light-harvesting complex II, predominant Chl a/b protein - MES 2-[N-Morpholino]ethanesulfonic acid - Pheo pheophytin - PS H photosystem II - Q_A bound plastoquinone, serving as the secondary electron acceptor in PS II (after Pheo) - SDS sodiumdodecylsulfate