Azide as a competitor of chloride in oxygen evolution by Photosystem II.

Oxygen evolution by higher plants requires chloride, which binds to a site associated with the oxygen-evolving complex of photosystem II (PSII). In this study, the inhibitory effect of the anion azide was characterized using steady state measurements of oxygen evolution activity in PSII-enriched thylakoid membranes. N3- (7.8 mM) inhibited O2 evolution activity by 50% when a standard buffer containing chloride was used. By considering Cl- as the substrate in O2 evolution assays, we found azide to be primarily competitive with Cl- with an inhibitor dissociation constant Ki of about 0.6 mM. An uncompetitive component with a Ki ' of 11 mM was also found. Removal of the 17 and 23 kDa polypeptides resulted in a decrease in each inhibition constant. A pH dependence study of O2 evolution activity showed that the pH maximum became narrower and shifted to a higher pH in the presence of azide. Analysis of the data indicated that an acidic residue defined the low side of the pH maximum with an apparent pKa of 6.7 in the presence of azide compared with 5.5 for the control. A basic residue was also affected, exhibiting an apparent pKa of 7.1 compared with a value of 7.6 for the control. This result can be explained by a simple model in which azide binding to the chloride site moves negative charge of the anion away from the basic residue and toward the acidic residue relative to chloride. As a competitor of chloride, azide may provide an interesting probe of the oxygen-evolving complex in future studies.     

Flash-induced proton transfer in photosynthetic bacteria

A proton electrochemical potential across the membranes of photosynthetic purple bacteria is established by a light-driven proton pump mechanism: the absorbed light in the reaction center initiates electron transfer which is coupled to the vectorial displacement of protons from the cytoplasm to the periplasm. The stoichiometry and kinetics of proton binding and release can be tracked directly by electric (glass electrodes), spectrophotometric (pH indicator dyes) and conductimetric techniques. The primary step in the formation of the transmembrane chemiosmotic potential is the uptake of two protons by the doubly reduced secondary quinone in the reaction center and the subsequent exchange of hydroquinol for quinone from the membrane quinone-pool. However, the proton binding associated with singly reduced promary and/or secondary quinones of the reaction center is substoichiometric, pH-dependent and its rate is electrostatically enhanced but not diffusion limited. Molecular details of protonation are discussed based on the crystallographic structure of the reaction center of purple bacteriaRb. sphaeroides andRps. viridis, structure-based molecular (electrostatic) calculations and mutagenesis directed at protonatable amino acids supposed to be involved in proton conduction pathways.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

Electrostatics and proton transfer in photosynthetic water oxidation

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).     

Measurement of photosynthetic oxygen evolution with a new type of oxygen sensor

We measured the light response curve of photosynthetic oxygen evolution by illuminating a leaf disc in an air-tight windowed chamber. Oxygen production was measured by monitoring the quenching of luminescence of an organometallic ruthenium compound. A photodiode based chlorophyll a fluorometer was used to measure the luminescence intensity. Oxygen evolution measurements with a traditional oxygen electrode gave the same numerical values at different light intensities when the same leaf disk was tested. The quality of the measurement signal of the new method was found to be similar to that obtained with the oxygen electrode method. The new luminescence based system is more stable against electrical disturbances than an oxygen electrode, its response to oxygen pressure changes is very rapid, and the new method allows the same basic equipment to be used for chlorophyll fluorescence and oxygen measurements.     

Ferrocyanide carbon-13 NMR line broadening as a probe of electron transfer reactions

The electron transfer reaction between ferrocyanide ion and the blue copper protein, stellacyanin, has been investigated by means of 13C NMR line broadening of the inorganic oxidant. The temperature dependence of the ferrocyanide line broadening gives an activation energy for the electron transfer reaction of 17 +/- 3 kJ. The apparent rate constant decreases with increasing concentration of K4Fe(CN)6, a result which can be explained either by formation of a strong precursor ferrocyanide--stellacyanin [Cu(II)] complex or by increased formation of KFe(CN)3-6 ion pairs. The direct electron transfer between ferrocyanide and ferricyanide has also been studied by 13C NMR line broadening of the former species. The ferricyanide concentration dependence of the exchange line broadening yields a value for the apparent second-order rate constant at 25 degrees C of k = 1.65 . 10(3) M-1 . s-1, in agreement with previously reported values derived from 14N NMR and isotope exchange studies. This rate constant shows a linear dependence on the K+ concentration, independent of ionic strength, a result which confirms the importance of ion pair species such as KFe(CN)3-6 and KFe(CN)2-6 in the direct electron transfer mechanism. The general applications of the method are discussed, including the considerations which suggest that a wide range of electron transfer rates, from about 1 s-1 to 4 . 10(3) s-1, are, in principle, accessible to this technique. The potential utility of ferrocyanide 13C spin--lattice relaxation time measurements is decreasing the lower limit of this range is also discussed.     

Azide as a probe of co-operative interactions in the mitochondrial F1-ATPase

(1) The hydrolytic activity of the isolated mitochondrial ATPase (F1) is strongly inhibited by azide. However, at very low ATP concentration (1 microM or less), no inhibition by azide is observed. (2) The azide-insensitive ATPase activity represents a high-affinity, low-capacity mode of turnover of F1. This is identified with the low Km, low Vmax component seen in steady-state kinetic studies in the absence of azide. (3) The azide-insensitive ATPase activity shows simple Michaelis-Menten kinetics, with Km = 3.2 microM, and Vmax = 1.1 mumol/min per mg (6 s-1). It is unaffected by anions such as sulphite, or by increasing pH in the range 7 to 8, both of which stimulate the maximal activity of F1. (4) Both the azide-insensitive and azide-sensitive components of F1-ATPase activity are equally inhibited by labelling the enzyme with 7-chloro-4-nitrobenzofurazan, by binding the natural inhibitor protein, or by cold denaturation of the enzyme. (5) It is concluded that azide-insensitive ATP hydrolysis represents catalysis by F1 involving a single catalytic site, and that azide acts by abolishing intersubunit cooperativity between the three catalytic sites of F1. Azide-sensitivity is thus a useful probe for events which affect the active site of F1 directly.     

Photoreaction of manganese catalyst in photosynthetic oxygen evolution

The formation of active O₂ evolving centers following addition of Mn²⁺ to Mn deficient Anacystis nidulans cells yielded an estimate of 6 to 12 Mn atoms associated with each O₂ evolving reaction center. Restoration of activity upon addition of Mn ions is affected in 3 ways: (1) Stimulation of the uptake of exogenous Mn into the cells—this uptake occurs in darkness, but is enhanced 5 to 10 fold by light; a high concentration of DCMU (1 × 10⁻⁵m) decreases this light enhanced influx no more than 50 to 75%; (2) Photoreactivation of the O₂ evolving centers, after excess Mn has been accumulated in the cells essentially no increase in Hill activity is observed unless the cells are illuminated. This photoreactivation is fully inhibited by 10⁻⁶m DCMU and partially by benzoquinone. The Q₁₀ of photoreactivation proper is close to 1; (3) Photoinhibition of the activation—photoreactivation occurs most effectively in weak intensities (< one-fiftieth photosynthetic saturation in normal cells). Apparently at higher intensities an inhibitory photoprocess is overriding. This inhibition proved reversible. The photoreactivation leads to new stable O₂ evolving centers as evidenced by an increase in the rate at saturating intensity, quantum yield, and the O₂ gush.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Calcium and photosynthetic oxygen evolution in cyanobacteria.

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca(2+). Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca(2+) addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca(2+) and Mg(2+) stimulate turnover, unit activation is Ca(2+) specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca(2+) -activated preparation. The Ca(2+) requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca(2+) of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605--609) these observations strongly suggest a site of Ca(2+) action within Photosystem II. The pronounced specificity of the Ca(2+) requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca(2+) dependence in P. luridum, French-press treatment is required for maximal expression of Ca(2+) -specific effects. French-press breakage causes a release of endogenous Ca(2+) from cells, supporting the view that added Ca(2+) restores oxygen evolution by satisfying a physiological requirement for the cation.     

Studies on a thermal reaction associated with photosynthetic oxygen evolution

The modulated polarographic technique of O₂ detection was applied to Chlorella to study the rate-limiting thermal reaction between Photosystem II and O₂ evolution. From an analysis of the operation of the polarograph at different frequencies, it was concluded that a first order thermal reaction of rate constant 305±20 (S.E.) s⁻¹ was consistent with the results of 22 °C. When the algae were successively studied in solutions made up with ²H₂O and H₂O, a kinetic isotopic effect for the rate constant of 1.29±0.05 (S.E.) was found. This suggests that the rate limiting step does not involve the breaking of the O-H bond in water. A temperature study of the rate constant indicated an activation energy of 5.9±0.5 (S.E.) kcal·mole⁻¹ and an entropy of activation of −25 cal·degree⁻¹·mole⁻¹. The linearity of the Arrhenius plot between 8 and 42 °C demonstrated that only one reaction was rate-limiting over this temperature range.     

Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K₃[Fe(CN)₆] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I₅₀ values were approx. 1–2, 4–6 and 0.04–0.10 M, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Q_p) which is also the electron donor for K₃ [Fe(CN)₆] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y - ANT2p- 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene - CCCP- carbonyl cyanide m-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl 6-isopropyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT- 2-iodo-6-isopropyl-3-methyl 2,4,4-trinitrodiphenyl ether - DPC- 1,5-diphenylcarbazide - DPIP- 2,6-dichlorophenolindophenol - FCCP- carbonyl cyanide p-trifuoromethoxyphenylhydrazone - FeCy- potassium ferricyanide - HQNO- 2-n-heptyl-4-hydroxyquinoline N-oxide - (MN)₄- the tetranuclear Mn cluster of water oxidizing complex - P680- photoactive Chl of the reaction center of Photosystem II - PCP- 2,3,4,5,6-pentachlorophenol - PS- photosystem - Q_A and Q_B- primary and secondary plastoquinones of PS II - Q_C and Q_Z- plastoquinone binding sites in the cytochrome blf complex - Q_p- membrane pool of plastoquinone - SiMo- sodium silicomolybdate - TMPD- N,N,N-tetramethyl-p-phenylenediamine - TTFB- 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - WOC- water oxidixing complex - Y_Z- tyrosine-161 of the Photosystem II D1 polypeptide     

Assay of photosynthetic oxygen evolution from single protoplasts

A semiquantitative assay for light-dependent O₂ evolution by a single mesophyll protoplast is described. The assay indicator is the density of aerotactic bacteria (Pseudomonas aeruginosa, ATCC 10145; `Engelmann experiment') attracted to the protoplast. Quantification is by dark field microphotometry. The sensitivity is about 50 femtomoles O₂ per protoplast per minute. The results demonstrate the biphasic nature of O₂ evolution of a single protoplast during photosynthetic induction. Computerized data acquisition yields traces which, until a steady state of photosynthetic O₂ evolution is reached, are identical to ordinary O₂ electrode traces.     

Flash-yield pattern for photosynthetic oxygen evolution in Chlorella and chloroplasts as a function of excitation intensity.

A study was carried out of the changes in flash-yield pattern for oxygen evolution at various light intensities. Oxygen measurements were made on algal and chloroplast samples, using a Joliot-type polarographic electrode. A mathematical model to describe the behavior of oxygen-evolving systems at various flash intensities was developed based on the binomial distribution. This model is capable of accurately predicting the oxygen flash-yield pattern over two orders of magnitude change in light intensity. The observed oxygen flash yields are accounted for at all flash intensities without changing the probability for misses and double hits by oxygen-evolving systems. It is concluded that intrinsic misses and double hits are observed, which are nearly independent of flash intensity. Also, the apparent optical cross section for oxygen evolution is found to increase as flash intensity decreases. It is suggested that inhomogeneity exists in the size of antenna-pigment aggregates associated with photosystem II reaction centers.     

Chlorophyll fluorescence transients of Photosystem II membrane particles as a tool for studying photosynthetic oxygen evolution

The rise of the chlorophyll fluorescence yield of Photosystem II (PS II) membranes as induced by high-intensity actinic light comprises only two distinct phases: (1) the initial O-J increase and (2) the subsequent J-P increase. Partial inhibition of the PS II donor side by heating or washing procedures which remove peripheral PS II proteins or cofactors of the oxygen-evolving complex results in decrease of magnitude and rate of the J-P phase. The rate constant of the J-P increase is directly proportional to the steady-state rate of oxygen evolution; complete suppression of the J-P phase corresponds to full inhibition. A characteristic dip after J-level is observed only in Tris-washed or severely heated PS II membranes; manganese release correlates with appearance of the dip after J-level as verified by EPR spectroscopy. Presence of stabilizing cosolutes (glycine betaine, sucrose) or addition of donor-side cofactors (bicarbonate, chloride, calcium) to PS II membranes before heating (47 °C, 5 min) diminishes J-P phase suppression and prevents dip appearance, whereas the addition after heating is without effect. In conclusion, analysis of chlorophyll fluorescence transients of PS II membranes is a potentially useful tool for investigations on photosynthetic oxygen evolution. A decreased rate of the J-P phase can be employed as a convenient indicator for partial inhibition of oxygen-evolution activity; the appearance of a dip after J-level is suggestive of manganese release. This revised version was published online in June 2006 with corrections to the Cover Date.