Force field development for cofactors in the photosystem II

We present a set of force field (FF) parameters compatible with the AMBER03 FF to describe five cofactors in photosystem II (PSII) of oxygenic photosynthetic organisms: plastoquinone‐9 (three redox forms), chlorophyll‐a, pheophytin‐a, heme‐b, and β‐carotene. The development of a reliable FF for these cofactors is an essential step for performing molecular dynamics simulations of PSII. Such simulations are important for the calculation of absorption spectrum and the further investigation of the electron and energy transfer processes. We have derived parameters for partial charges, bonds, angles, and dihedral‐angles from solid theoretical models using systematic quantum mechanics (QM) calculations. We have shown that the developed FF parameters are in good agreement with both ab initio QM and experimental structural data in small molecule crystals as well as protein complexes. © 2012 Wiley Periodicals, Inc.     

Redox potentials of chlorophylls and beta-carotene in the antenna complexes of photosystem II

Electron transfer (ET) processes in reaction centers (RC) of photosystem II (PSII) are prerequisites of oxygen generation. They are promoted by energy transfer from antenna to RC. Here, we calculated the redox potentials of chlorophylla/beta-carotene (Chla/Car) in PSII CP43/CP47 antenna complexes, solving the linearized Poisson-Boltzmann (LPB) equation based on the PSII crystal structure. The majority of antenna Chla redox potentials for reduction/oxidation were lower than those of RC Chla. Hence, ET events with excess electrons remain localized in the RC. Simultaneously antenna Chla can serve as an efficient cation sink to rereduce RC Chla if normal PSII function is inhibited. Especially three antenna Chla (Chl-47, Chl-18, and Chl-12) and two Car bridging the space between Chl(Z(D1)) and cytochrome (cyt) b559 have the same level of oxidation redox potential. Together with Chl(Z(D2)) they form an electron hole transfer pathway and temporary storage device guiding from the oxidized P680(+.) Chla to the cyt b559. This path may play a photoprotective role as efficient electron hole quencher.     

Redox state of the photosynthetic electron transport chain in wild-type and mutant leaves of Arabidopsis thaliana: Impact on photosystem II fluorescence

In addition to the photosynthetic linear electron transport, several alternative electron transport routes exist in thylakoids of higher plants. The plastoquinone (PQ) pool acts as a common electron carrier in these pathways. In the cyclic electron flow around photosystem I (PSI), reduced ferredoxin is used by the ferredoxin-quinone reductase (FQR) to reduce the PQ pool. Chlororespiratory pathway consists in the reduction of the PQ pool by the NAD(P)H dehydrogenase (NDH). These alternative pathways and their role in photosynthesis are still not fully understood. In the present study, the accumulation kinetics of quinone acceptors was measured by fluorescence induction in leaves of Arabidopsis thaliana wild-type and mutants altered in alternative electron pathways after various light- and dark-adaptation conditions. Results show that NDH activity can be probed by fluorescence induction during light-to-dark transition of plants. Also, the activity of FQR pathway did not affect directly the FI kinetics. However, the accumulation kinetics of reduced PQ under actinic light was dependant on the redox state of PSI acceptors prior to illumination.     

NO reductase activity of the tetraheme cytochrome C554 of Nitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase (HAO). We show here that cyt c(554) also has significant NO reductase activity. The protein contains one high-spin and three low-spin c-type hemes. HAO catalyzed reduction of the cyt c(554), ligand binding, intermolecular electron transfer, and kinetics of NO reduction by cyt c(554) have been investigated. We detect the formation of a NO-bound ferrous heme species in cyt c(554) by EPR and M?ssbauer spectroscopies during the HAO catalyzed oxidation of hydroxylamine, indicating that N-oxide intermediates produced from HAO readily bind to cyt c(554). In the half-reduced state of cyt c(554), we detect a spin interaction between the [FeNO](7) state of heme 2 and the low-spin ferric state of heme 4. We find that ferrous cyt c(554) will reduce NO at a rate greater than 16 s(-1), which is comparable to rates of other known NO reductases. Carbon monoxide or nitrite are shown not to bind to the reduced protein, and previous results indicate the reactions with O(2) are slow and that a variety of ligands will not bind in the oxidized state. Thus, the enzymatic site is highly selective for NO. The NO reductase activity of cyt c(554) may be important during ammonia oxidation in N. europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine.     

Effects of (60)Co gamma radiation on thylakoid membrane functions in Anacystis nidulans

In photosynthetic organisms oxidative stress is known to result in photoinactivation of photosynthetic machinery. We investigated effects of ⁶⁰Co γ radiation, which generates oxidative stress, on thylakoid structure and function in cyanobacteria. Cells of unicellular, non-nitrogen fixing cyanobacterium Anacystis nidulans (Synechococcus sp.) showed D₁₀ value of 257 Gy of ⁶⁰Co γ radiation. When measured immediately after exposure, cells irradiated with 1500 Gy (lethal dose) of ⁶⁰Co γ radiation did not show any differences in photosynthetic functions such as CO₂ fixation, O₂ evolution and partial reactions of photosynthetic electron transport in comparison to unirradiated cells. Incubation of irradiated cells for 24 h in light or dark resulted in decline in photosynthesis. The decline in photosynthesis was higher in the cells incubated in light as compared to the cells incubated in dark. Among the partial reactions of electron transport, only PSII activity declined drastically after incubation of irradiated samples. This was also supported by the analysis of membrane functions using thermoluminescence. Exposure of cyanobacteria to high doses of ⁶⁰Co γ radiation did not affect the thylakoid membrane ultrastructure immediately after exposure as shown by electron microscopy. The level of reactive oxygen species (ROS) in irradiated cells was 20 times higher as compared to control. In irradiated cells de novo protein synthesis was reduced considerably immediately after irradiation. Treatment of cells with tetracycline also affected photosynthesis as in irradiated cells. The results showed that photoinhibition of photosynthetic apparatus after incubation of irradiated cells was probably augmented due to reduced protein synthesis. Active photosynthesis is known to require uninterrupted replenishment of some of the proteins involved in electron transport chain. The defective thylakoid membrane biogenesis may be leading to photosynthetic decline post-irradiation.     

Direct electrochemistry of cytochrome c on nanotextured diamond surface

Nanotextured diamond surfaces with geometrical properties close to protein dimensions were used for the realization of direct electron transfer of cytochrome c (cyt c) without any covalent bonding. The peroxidase activity of native and denatured cyt c was also investigated. Cyclic voltammograms of native cyt c show quasi-reversible electron transfer reactions, while no heme redox activity is detected for denatured cyt c. Unfolding (denaturation) of cyt c can be achieved in the presence of hydrogen peroxide. Partially or fully denatured cyt c showed higher peroxidase activity than native cyt c. This is because denatured cyt c loses its tertiary structure and hydrogen peroxide is easier to access the heme redox center. The apparent Michaelis–Menten constant K_m for native and denatured cyt c has been determined to be 0.23 mM and 0.08 mM.     

Control of quinone redox potentials in photosystem II: Electron transfer and photoprotection

In O(2)-evolving complex Photosystem II (PSII), an unimpeded transfer of electrons from the primary quinone (Q(A)) to the secondary quinone (Q(B)) is essential for the efficiency of photosynthesis. Recent PSII crystal structures revealed the protein environment of the Q(A/B) binding sites. We calculated the plastoquinone (Q(A/B)) redox potentials (E(m)) for one-electron reduction with a full account of the PSII protein environment. We found two different H-bond patterns involving Q(A) and D2-Thr217, resulting in an upshift of E(m)(Q(A)) by 100 mV if the H bond between Q(A) and Thr is present. The formation of this H bond to Q(A) may be the origin of a photoprotection mechanism, which is under debate. At the Q(B) side, the formation of a H bond between D2-Ser264 and Q(B) depends on the protonation state of D1-His252. Q(B) adopts the high-potential form if the H bond to Ser is present. Conservation of this residue and H-bond pattern for Q(B) sites among bacterial photosynthetic reaction centers (bRC) and PSII strongly indicates their essential requirement for electron transfer function.     

OXYGEN MEDIATED PHOTOSYSTEM I ACTIVITY IN THYLAKOID MEMBRANES MONITORED BY A PHOTOELECTROCHEMICAL CELL

Abstract— A photoelectrochemical cell has been used to monitor the effects of three enzymes on the photocurrent produced by isolated spinach thylakoids. The enzymes were glucose oxidase, superoxide dismutase and catalase. It is shown that all three inhibit the photocurrent to varying degrees. The results demonstrate that electron transport to the working electrode is mediated by oxygen. Further, the activity monitored originated from photosystem I with oxygen as the acceptor and photosystem II/plastoquinone as the donor. Thus, the photoelectrochemical cell constitutes a potential new approach for the monitoring of pseudocyclic electron transport.     

Involvement of reactive oxygen species in the UV-B damage to the cyanobacterium Anabaena sp

Reactive oxygen species (ROS) are involved the damage of living organisms under environmental stress including UV radiation. Cyanobacteria, photoautotrophic prokaryotic organisms, also suffer from increasing UV-B due to the depletion of the stratospheric ozone layer. The increased UV-B induces the production of ROS in vivo detected by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Ascorbic acid and N-acetyl-L-cysteine (NAC) scavenged ROS effectively, while alpha-tocopherol acetate or pyrrolidine dithiocarbamate (PDTC) did not. The presence of rose bengal and hypocrellin A increased the ROS level by photodynamic action in the visible light. The presence of the herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), increased ROS production slightly, and ROS formation was greatly enhanced by the addition of methyl viologen due to the fact that this redox system diverts electrons from PSI to oxygen and thus forms ROS. UV-B induces ROS generation by photodynamic action and inhibition of the electron transport by damaging the electron receptors or enzymes associated with the electron transport chain during photosynthesis.     

POLYPHASIC CHLOROPHYLL a FLUORESCENCE TRANSIENT IN PLANTS AND CYANOBACTERIA*

Abstract— The variable chlorophyll (Chl) a fluorescence yield is known to be related to the photochemical activity of photosystem II (PSII) of oxygen-evolving organisms. The kinetics of the fluorescence rise from the minimum yield, F₀, to the maximum yield, F_m, is a monitor of the accumulation of net reduced primary bound plastoquinone (Q_A) with time in all the PSII centers. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which allows data accumulation over several orders of magnitude of time (40 μs to 120 s), we have measured on a logarithmic time scale, for the first time, the complete polyphasic fluorescence rise for a variety of oxygenic plants and cyanobacteria at different light intensities. With increasing light intensity, the fluorescence rise is changed from a typical O-I-P characteristic to curves with two intermediate levels J and I, both of which show saturation at high light intensity but different intensity dependence. Under physiological conditions, Chl a fluorescence transients of all the organisms examined follow the sequence of O-J-I-P. The characteristics of the kinetics with respect to light intensity and temperature suggest that the O-J phase is the photochemical phase, leading to the reduction of Q_A to Q_A^-. The intermediate level I is suggested to be related to a heterogeneity in the filling up of the plastoquinone pool. The P is reached when all the plastoquinone (PQ) molecules are reduced to PQH₂. The addition of 3-(3–4-dichlorophenyl)-1,1-dimethylurea leads to a transformation of the O-J-I-P rise into an O-J rise. The kinetics of O-J-I-P observed here was found to be similar to that of O-I₁-I₂-P, reported by Neubauer and Schreiber (Z. Naturforsch. 42c , 1246–1254, 1987). The biochemical significance of the fluorescence steps O-J-I-P with respect to the filling up of the plastoquinone pool by PSII reactions is discussed.     

Light induced oxidative water splitting in photosynthesis: energetics, kinetics and mechanism

The essential steps of photosynthetic water splitting take place in Photosystem II (PSII) and comprise three different reaction sequences: (i) light induced formation of the radical pair P680(+)Q(A)(-), (ii) P680(+) driven oxidative water splitting into O(2) and four protons, and (iii) two step plastoquinone reduction to plastoquinol by Q(A)(-). This mini-review briefly summarizes our state of knowledge on energetics, kinetics and mechanism of oxidative water splitting. Essential features of the two types of reactions involved are described: (a) P680(+) reduction by the redox active tyrosine Y(z) and (b) sequence of oxidation steps induced by Y(z)(ox) in the water-oxidizing complex (WOC). The rate of the former reaction is limited by the non-adiabatic electron transfer (NET) step and the multi-phase kinetics shown to originate from a sequence of relaxation processes. In marked contrast, the rate of the stepwise oxidation by Y(z)(ox) of the WOC up to the redox level S(3) is not limited by NET but by trigger reactions which probably comprise proton shifts and/or conformational changes. The overall rate of the final reaction sequence leading to formation and release of O(2) is assumed to be limited by the electron transfer step from the S(3) state of WOC to Y(z)(ox) due to involvement of an endergonic redox equilibrium. Currently discussed controversial ideas on possible pathways are briefly outlined. Several crucial points of the mechanism of oxidative water splitting, like O-O bond formation, role of local proton shift(s), details of hydrogen bonding, are still not clarified and remain a challenging topic of future research.     

REDOX PROTEINS AS ELECTRON ACCEPTORS FROM CHLOROPHYLL TRIPLET STATE IN LIPID BILAYER VESICLES: KINETICS OF REDUCTION OF MEMBRANE RECONSTITUTED CYTOCHROME c OXIDASE MEDIATED BY 2,5-DI-t-BUTYL BENZOQUINONE AND CYTOCHROME c

Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition.     

Effect of Ce~(3+) on spectral characteristic of D1/D2/Cytb559 complex from spinach

In our early researches, lanthanum and cerium could enter plant and bind to porphyrin of chlorophyll to form Ln3+-chllorophyll. La and Ce greatly increase photosystem II (PSII) activity and PSII electron transport rate, and the fluorescence emission peaks of PSII are blue-shifted [1—4]. Do REEs coordinate with PSII reaction center complex in vivo? Moreover, do REEs coordinate with D1(30 kD)/D2(32 kD)/Cytb559 (~9 kD) reaction center complex of site of producing pri-mary reaction-p…     

Protein film electrochemistry of microsomes genetically enriched in human cytochrome p450 monooxygenases

This communication demonstrates direct electron delivery from electrodes to cyt P450 reductases in stable films ( approximately 100 nm thick) of genetically enriched CYP1A2 and CYP3A4 microsomes made by layer-by-layer assembly with polyions. Reversible voltammetry of films containing genetically enriched cyt P450 monooxygenase microsomes was shown to involve cyt P450 reductase by comparison with the pure rabbit reductase and by lack of characteristic reactions of iron heme enzymes, such as reaction of the FeII form with CO and catalytic electrochemical reduction of oxygen and hydrogen peroxide. The microsome films were activated electrochemically to catalyze styrene epoxidation, consistent with the pathway utilized in the human liver, although further work is required to establish this definitively.     

Kinetic performance and energy profile in a roller coaster electron transfer chain: a study of modified tetraheme-reaction center constructs

In many electron-transfer proteins, the arrangement of cofactors implies a succession of uphill and downhill steps. The kinetic implications of such arrangements are examined in the present work, based on a study of chimeric photosynthetic reaction centers obtained by expressing the tetraheme subunit from Blastochloris viridis in another purple bacterium, Rubrivivax gelatinosus. Site-directed mutations of the environment of heme c559, which is the immediate electron donor to the primary donor P, induced modifications of this heme's midpoint potential over a range of 400 mV. This resulted in shifts of the apparent midpoint potentials of the neighboring carriers, yielding estimates of the interactions between redox centers. At both extremities of the explored range, the energy profile of the electron-transfer chain presented an additional uphill step, either downstream or upstream from c559. These modifications caused conspicuous changes of the electron-transfer rate across the tetraheme subunit, which became approximately 100-fold slower in the mutants where the midpoint potential of c559 was lowest. A theoretical analysis of the kinetics is presented, predicting a displacement of the rate-limiting step when lowering the potential of c559. A reasonable agreement with the data was obtained when combining this treatment with the rates predicted by electron transfer theory for the individual rate constants.     

Relationship between the structural and functional changes of the photosynthetic apparatus during chloroplast-chromoplast transition in flower bud of Lilium longiflorum

The relationship between the structural and functional changes of the photosynthetic apparatus in the flower bud of Lilium longiflorum during chloroplast-chromoplast transition was examined. Compared with green petals, there was a five-fold increase of the carotenoid content in yellow petals, whereas the chlorophyll content decreased five-fold. Absorption and emission fluorescence spectra of chromoplasts indicated that newly synthesized carotenoids were not associated with photosystem II (PSII) photochemistry. The maximum quantum yield in the remaining PSII reaction centers remained constant during the chromoplast formation, whereas the photosynthetic electron transport beyond PSII became inhibited, as indicated by a marked decrease of the O2 evolution capacity, of the photochemical quenching of chlorophyll-alpha fluorescence and of the operational quantum yield of photosynthetic electron transport. Deconvoluted fluorescence emission spectra indicated a more rapid degradation of photosystem I (PSI) complexes than of PSII during chromoplast formation. Compared with green petals, the spillover between PSII and PSI decreased by approximately 40% in yellow petals. Our results indicate that during chloroplast-chromoplast transition in the flower bud of L. longiflorum, PSII integrity was preserved longer than the rest of the photosynthetic apparatus.     

Structure–Function of the Cytochrome b6f Complex†

The structure and function of the cytochrome b₆ f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme‐binding transmembrane domain of the cyt b polypeptide between b₆ f and bc₁ complexes, and the rubredoxin‐like membrane proximal domain of the Rieske [2Fe‐2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme c_n, not found in the bc₁ complex, but electronically tightly coupled to the heme b_n of the b₆ f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl‐stigmatellin, show the latter to be ligands of heme c_n, implicating heme c_n as an n‐side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe‐2S] protein between the membrane‐bound PQH₂ electron/H⁺ donor and the cytochrome f acceptor to complete the p‐side electron transfer circuit; (ii) PQ/PQH₂ between n‐ and p‐sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p‐side [2Fe‐2S] niche; (b) the role of the n‐side of the b₆ f complex and heme c_n in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b₆ f complex, and of heme c_n in the firmicute bc complex suggests the concept that hemes b_n‐c_n define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc₁ complex.     

Electrochemically Gated Long‐Distance Charge Transport in Photosystem I

The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to understanding charge transport mechanisms and to the design of bioelectronic sensors and solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different protein sites. Herein, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi level and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes. Current–distance measurements at different potentiostatic conditions indicate that PSI supports long‐distance transport that is electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.     

Resonance Raman spectroscopy of nitric oxide reductase and cbb(3) heme-copper oxidase

Elucidating the structure and properties of the active sites in cbb3 heme-copper oxidase and in nitric oxide reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b3-CuB center of cbb3 heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b3-FeB center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b3 bond intact, and reformation of the heme b3-O-FeB dinuclear center causes the rupture of the proximal His-Fe heme b3 bond. In the reactions of Nor and cbb3 with O2, distinct oxidized heme b3 species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O2 and NO by cbb3 oxidase and nitric oxide reductase are compared and discussed.