Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo^–) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 M) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 M and 2 M resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the variable part of chlorophyll fluorescence (which is the recombination luminescence of the pair P₆₈₀ ⁺ Pheo^–). A 50% inhibition was induced by 5 M K-15 and the effect was maximal in the range 20 to 200 M. Photooxidation of P₆₈₀ in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 M). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo^– and P₆₈₀ ⁺ (or Z⁺) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for Q_A missing in the preparation.At a concentration of 0.5–50 M K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl₂. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo^– to cytochrome b559.Abbreviations K-15 4-[methoxy-bis-(trifluoromethyl)methyl]-2,6-dinitrophenyl hydrazone methyl ketone - P₆₈₀ the primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A and Q_B the primary and the secondary electron acceptor of PS II - RC reaction centre - SiMo silicomolybdate - TMPD N,N,N,,N,-tetramethyl-p-phenylenediamine - Z secondary electron donor of PS II     

Effects of hydrostatic pressure on the kinetics of electron transfer in an isolated system of chloroplast cytochrome bf complex,plastocyanin and P700

The effects of pressure on the kinetics of redox reactions in and around the chloroplast cytochrome bf complex were studied using a reconstituted system consisting of Photosystem I (PS I) particles, cytochrome bf complex and plastocyanin (PC), all derived from pea chloroplasts. There were no significant permanent effects of pressure in the range 0.1–191 MPa on the reaction kinetics, or on the shape of the absorption spectra of components studied. Discernable effects on rate-coefficients of increasing pressure were observed on the reduction of P700⁺ by PC^I, on the reduction of PC^II by ascorbate, and on the oxidation of decyl plastoquinol by the bf complex. The volumes of activation ΔV^# were determined from the dependence of the rate-coefficient on pressure using: $$(\partial lnk/\partial P)_T = - \Delta V^\# /RT.$$ The volume of activation is the difference in partial molar volume between the activated state and the reactants for the redox reaction. Such data was sought to help define in detail those redox reactions and the corresponding activated states. For the reduction of P700⁺ by PC^I and the oxidation of decyl plastoquinol by the bf complex, the rate coefficient decreased with increase in pressure, whilst for the reduction of PC^II by ascorbate it increased. The corresponding volumes of activation were 9.6±0.6×10^-6 m³ mol^-1, 18±2×10^-6 m³ mol^-1 and -14±1×10^-6 m³ mol^-1, respectively. Much of the pressure-dependence of PC^II reduction by ascorbate was ascribed to an increase in ascorbate ionisation with increase in pressure. There was little effect of pressure on the kinetics of oxidation of ferrocytochrome f by PC^II, or on the equilibrium constant of the redox pair ferrocytochrome f/ferricytochrome f: PC^II/PC^I. Possible physical bases for these activation volumes are discussed, and they are compared with literature values.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain)

A three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the "best" structure in terms of charges, interactions, and complementarity of the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 A, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 A2.     

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.     

On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codon-correct cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b ₆  f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b ₆ , and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b ₆ and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b ₆ and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed. Received: 18 September 1996 / Accepted: 15 October 1996     

Electron transfer through the isolated mitochondrial cytochrome b-c1 complex

(1) A kinetic analysis of electron donation into and through the cytochrome b-c₁ complex isolated from bovine heart mitochondria has been undertaken, using trimethylquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Probing the events of photoinhibition by altering electron-transport activity and light-harvesting capacity in chloroplast thylakoids

Abstract. The effect of photoinhibition on the activity of photosystem II (PSII) in spinach chloroplasts was investigated. Direct light-induced absorbance change measurements at 320 nm (Δ A ₃₂₀) provided a measure of the PSII charge separation reaction and revealed that photoinhibition prevented the stable photoreduction of the primary quinone acceptor Q_A. Sensitivity to photoinhibition was substantially enhanced by treatment of thylakoids with NH₂OH which extracts manganese from the H₂O-splitting enzyme and prevents electron donation to the reaction centre. Incubation with 3-(3,4,-dichlorophenyl)-1,1-dimethylurea (DCMU) during light exposure did not affect the extent of photoinhibitory damage. The chlorophyll (Chl) b -less chlorina (2 mutant of barley displayed a significantly smaller light-harvesting antenna size of PSII (about 20% of that in wild type chloroplasts) and, simultaneously, a lower sensitivity to photoinhibition. These observations suggest that photoinhibition depends on the amount of light absorbed by PSII and that the process of photoinhibition is accelerated when electron donation to the reaction centre is prevented. It is postulated that the probability of photoinhibition is greater when excitation energy is trapped by P680⁺, the oxidized form of the PSII reaction centre. The results are discussed in terms of the D1/D2 heterodimer which contains the functional PSII components P680, pheophytin, Q_A and Q_B.     

A new approach to experimental investigation of dynamic self-organization in reaction centers of purple bacteria

The results of a study of molecular self-organization processes in the reaction centers (RC) ofRb. Sphaeroides purple bacteria by the method of pulsed optical excitation is presented. The existence of a bistability domain for the parameters of RC recovery kinetics is shown. A good agreement between the theory and experimental results is obtained.     

Molecular dynamics simulations of the cytochrome c3-rubredoxin complex from Desulfovibrio vulgaris.

Molecular dynamics simulations have been carried out on the complex formed between the tetraheme cytochrome c3 and the iron protein rubredoxin from the sulfate-reducing bacterium Desulfovibrio vulgaris. These simulations were performed both with explicit solvent water molecules included, and without solvent molecules using a distance-dependent dielectric constant to approximate the screening effects of solvent. The results of both simulations are strikingly different, indicating that the representation of environmental effects is important in such simulations. For example, a striking adaptation of the two proteins seen in the nonsolvated simulation is not seen when explicit solvent water is included; in fact, the complex appears to become weaker in the solvated simulation. Nonetheless, the iron-iron distance decreases more significantly in the solvated simulation than in the nonsolvated simulation. It was found that in both cases molecular dynamics optimized the structures further than energy minimization alone.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

A Nonadiabatic Theory for Ultrafast Catalytic Electron Transfer: A Model for the Photosynthetic Reaction Center

A non-adiabatic theory of Electron Transfer (ET), which improves the standard theory near the inversion point and becomes equivalent to it far from the inversion point, is presented. The complex amplitudes of the electronic wavefunctions at different sites are used as Kramers variables for describing the quantum tunneling of the electron in the deformable potential generated by its environment (nonadiabaticity) which is modeled as a harmonic classical thermal bath. After exact elimination of the bath, the effective electron dynamics is described by a discrete nonlinear Schrödinger equation with norm preserving dissipative terms and a Langevin random force, with a frequency cut-off, due to the thermalized phonons. This theory reveals the existence of a specially interesting marginal case when the linear and nonlinear coefficients of a two electronic states system are appropriately tuned for forming a Coherent Electron-Phonon Oscillator (CEPO). An electron injected on one of the electronic states of a CEPO generates large amplitude charge oscillations (even at zero temperature) associated with coherent phonon oscillations and electronic level oscillations. This fluctuating electronic level may resonate with a third site which captures the electron so that Ultrafast Electron Transfer (UFET) becomes possible. Numerical results are shown where two weakly interacting sites, a donor and a catalyst, form a CEPO that triggers an UFET to an acceptor. Without a catalytic site, a very large energy barrier prevents any direct ET. This UFET is shown to have many qualitative features similar to those observed in the primary charge separation in photosynthetic reaction centers. We suggest that more generally, CEPO could be a paradigm for understanding many selective chemical reactions involving electron transfer in biosystems.     

A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.     

Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Comparison of low oxidoreduction potential cytochrome c553 from Desulfovibrio vulgaris with the class I cytochrome c family

The cytochrome c553 from Desulfovibrio vulgaris (DvH c553) is of importance in the understanding of the relationship of structure and function of cytochrome c due to its lack of sequence homology with other cytochromes, and its abnormally low oxido-reduction potential. In evolutionary terms, this protein also represents an important reference point for the understanding of both bacterial and mitochondrial cytochromes c. Using the recently determined nuclear magnetic resonance (NMR) structure of the reduced protein we compare the structural, dynamic, and functional characteristics of DvH c553 with members of both the mitochondrial and bacterial cytochromes c to characterize the protein in the context of the cytochrome c family, and to understand better the control of oxido-reduction potential in electron transfer proteins. Despite the low sequence homology, striking structural similarities between this protein and representatives of both eukaryotic [cytochrome c from tuna (tuna c)] and prokaryotic [Pseudomonas aeruginosa c551 (Psa c551)] cytochromes c have been recognized. The previously observed helical core is also found in the DvH c553. The structural framework and hydrogen bonding network of the DvH c553 is most similar to that of the tuna c, with the exception of an insertion loop of 24 residues closing the heme pocket and protecting the propionates, which is absent in the DvH c553. In contrast, the Psa c551 protects the propionates from the solvent principally by extending the methionine ligand arm. The electrostatic distribution at the recognized encounter surface around the heme in the mitochondrial cytochrome is reproduced in the DvH c553, and corresponding hydrogen bonding networks, particularly in the vicinity of the heme cleft, exist in both molecules. Thus, although the cytochrome DvH c553 exhibits higher primary sequence homology to other bacterial cytochromes c, the structural and physical homology is significantly greater with respect to the mitochondrial cytochrome c. The major structural and functional difference is the absence of solvent protection for the heme, differentiating this cytochrome from both reference cytochromes, which have evolved different mechanisms to cover the propionates. This suggests that the abnormal redox potential of the DvH c553 is linked to the raised accessibility of the heme and supports the theory that redox potential in cytochromes is controlled by heme propionate solvent accessibility.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.