The cationic plastoquinone radical of the chloroplast water splitting complex: Hyperfine splitting from a single methyl group determines the EPR spectral shape of Signal II

The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH₃ group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A_⊥ = 27.2 MHz and $\text{A}{}_{\mathrm{}}\text{= 31.4}\text{MHz}$. The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH₃ bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane.     

On the Molecular Identity of ESR Signal II Observed in Photosynthetic Systems: The Effect of Heptane Extraction and Reconstitution With Plastoquinone and Deuterated Plastoquinone

Speculation as to the identity of Signal II, the light-induced, broad, slow decaying electron spin resonance signal with hyperfine structure observed in photosynthetic materials, has tended to center on the semiquinone of plastoquinone. Experiments reported here were designed to give direct evidence bearing on that speculation. Heptane extraction of lipids from lyophilized spinach and tobacco chloroplast fragments reduced the amplitude of Signal II and increased the ratio of Signal I:Signal II. Reconstitution of the system by the addition of plastoquinone partially restored Signal II as well as the ratio of Signal I:Signal II to its pre-extraction condition. Addition of totally deuterated plastoquinone to extracted chloroplasts in which considerable Signal II had survived heptane extraction resulted in a spectrum which showed the characteristic hallmarks of Signal II observed in totally deuterated organisms. These results establish that a free radical immediately derived from plastoquinone contributes to Signal II. The data taken by themselves are consistent with plastochromanoxyl as well as plastosemiquinone free radicals giving rise to Signal II. Other contributors to Signal II are not ruled out.     

Immobilized radicals. IV. Biological semiquinone anions and neutral semiquinones

The semiquinone anion and neutral semiquinone radicals of benzoquinone, vitamin K-1, ubiquinone and plastoquinone were generated in both protic and aprotic solvents and frozen to produce immobilized spectra. The linewidths of the neutral semiquinones were always much larger than those of the corresponding anion radicals. Furthermore, the spectra of the neutral radicals often exhibit fine structure. When compared with in vivo spectra of semiquinones, these model systems suggest that the ubisemiquinone anion radical observed in photosynthetic bacteria can exist in either a protic or aprotic environment. There is also the implication that Signal II in chloroplasts may be a plastosemiquinone radical with a spin distribution similar to that of the neutral radical.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

Supposition of the origin of Signal II from random and oriented chloroplasts

From previous studies of biological semiquinones in different solvents, the origin of Signal II in chloroplasts is hypothesized to be a plastosemiquinone anion radical perturbed by a metal cation. Assuming this model, theoretical principal g factors and hyperfine splitting constants were calculated and used to simulate the random spectrum of spinach Signal II. Oriented chloroplasts were used to determine the principal angles of this model. Oriented chloroplasts from collard greens showed a different angular dependency of Signal II from those of spinach as well as the presence of added fine structure.     

The structure of the secondary donor of Photosystem II investigated by EPR at 9 and 35 GHz

Signal II of plant photosynthesis, which is generally thought to be connected to the secondary donor complex of Photosystem II, has been investigated with EPR spectroscopy at 9 and 35 GHz. From the spectrum at 35 GHz of deuterated Chlorella vulgaris, the principle values of the g-tensor are determined to be g_xx = 2.0074, g_yy = 2.0044 and g_zz = 2.0023. Proton hyperfine coupling tensor elements and orientations were determined from spectral simulation of random and oriented samples, assuming that Signal II is due to a plastose-miquinone cation having its π-electrons in an antisymmetric orbital as proposed by P.J. O'Malley, G.T. Babcock and R.C. Prince (Biochim. Biophys. Acta 765 (1984) 370–379). In contrast to their work, it is found that most hyperfine interaction is due to the methylene group at ring position 5 and to both hydroxyl groups. One of the hydroxyl groups shows bond bending of 35°. We presume that this is due to hydrogen bonding and that this bond stabilizes the antisymmetric orbital of the π-electrons.     

Similarity of EPR Signal IIf rise and P-680+ decay kinetics in Tris-washed chloroplast Photosystem II preparations as a function of pH

The rise time, of Signal II_f and the decay time of P-680⁺ have been measured kinetically as a function of pH by using EPR. The Photosystem II-enriched preparations which were used as samples were derived from spinach chloroplasts, and they evolved oxygen before Tris washing. The onset kinetics of Signal II_f are in agreement, within experimental error, with the fast component of the decay of an EPR signal attributable to P-680⁺. The signal II_f rise kinetics also show good agreement with published values of the pH dependence of the decay of P-680⁺ measured optically (Conjeaud, H. and Mathis, P. (1980) Biochim. Biophys. Acta 590, 353–359). These results are consistent with a model where the species Z (or D₁) responsible for Signal II_f is the immediate electron donor to P-680⁺ in tris-washed Photosystem II fragments.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.     

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II_slow and Signal II_fast of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y_Z and Y_D of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043 ± 0.0003 and a line width of 10 ± 1 G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q_A in the absence of magnetic interaction with the non-heme Fe²⁺. We conclude that peroxynitrite impairs PSII electron transport in the Q_AFe²⁺ niche.     

An EPR study of Signal II in oriented photosystem II membranes

Signal II of plant photosynthesis, which is thought to be due to a plastosemiquinone cation radical, has been studied by EPR at 9 and 35 GHz in non-oriented and partly oriented PS II particles. The spectra measured of the oriented particles at 35 GHz show that the molecular Z-axis, which is the axis perpendicular to the plane of the radical, makes an angle of 60° with the membrane normal. All spectra could be computer-simulated with one set of parameters. This set is essentially the same as that given earlier on the basis of EPR spectroscopy on non-oriented membranes [(1985) Biochim. Biophys. Acta 809, 421-428], except that the bond bending of the hydroxyl group on ring position 1 is found to be 60°, resulting in a somewhat smaller isotropie hyperfine splitting of the hydroxyl proton.Signal IIEPROrientationHyperfine coupling     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Programmed cell death in plants: Protective effect of mitochondrial-targeted quinones

Ubiquinone or plastoquinone covalently linked to synthetic decyltriphenylphosphonium (DTPP⁺) or rhodamine cations prevent programmed cell death (PCD) in pea leaf epidermis induced by chitosan or CN⁻. PCD was monitored by recording the destruction of cell nuclei. CN⁻ induced the destruction of nuclei in both epidermal cells (EC) and guard cells (GC), whereas chitosan destroyed nuclei in EC not in GC. The half-maximum concentrations for the protective effects of the quinone derivatives were within the pico- and nanomolar range. The protective effect of the quinones was removed by a protonophoric uncoupler and reduced by tetraphenylphosphonium cations. CN⁻-Induced PCD was accelerated by the tested quinone derivatives at concentrations above 10⁻⁸–10⁻⁷ M. Unlike plastoquinone linked to the rhodamine cation (SkQR1), DTPP⁺ derivatives of quinones suppressed menadione-induced H₂O₂ generation in the cells. The CN⁻-induced destruction of GC nuclei was prevented by DTPP⁺ derivatives in the dark not in the light. SkQR1 inhibited this process both in the dark and in the light, and its effect in the light was similar to that of rhodamine 6G. The data on the protective effect of cationic quinone derivatives indicate that mitochondria are involved in PCD in plants.     

Endor characterization and D2O exchange in the \begin{array}{*{20}c} {\mathop Z\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array} /{\kern 1pt} \begin{array}{*{20}c} {\mathop D\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array} radical in photosystem II

The early suggestion by Lozier and Butler (Photochem. Photobiol. 17, 133–137 (1973)) that EPR Signal II arises from radicals associated with the water-splitting process in PSII has been confirmed and extended over the intervening years. Recent work has identified the Signal II radicals, \(\begin{array}{*{20}c} {\mathop D\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array}\) and \(\begin{array}{*{20}c} {\mathop Z\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array}\) , with plastosemiquinone cation species. In the experiments presented here we have used ENDOR spectroscopy and D₂O/H₂O exchange to characterize these paramagnets in more detail. The ENDOR matrix region, which arises from protons which interact weakly with the unpaired electron spin, is well-resolved at 4 K and at least seven resonances are apparent. A number of hyperfine couplings in the 3–8 MHz range are observed and are suggested to arise from methyl or hydroxyl protons which occur as substituents on the plastosemiquinone cation ring or from amino acid protons hydrogen-bonded to the 1,4-hydroxyl groups. Orientation selection experiments are consistent with these possibilities. D₂O/H₂O exchange shows that the D⁺/Z⁺ site is accessible to solvent. However, the exchange occurs slowly and is not complete even after 72 hours which suggests that the free radicals are functionally isolated from solvent water.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Long-range interactions of <Emphasis Type="Italic">Chara</Emphasis> chloroplasts are sensitive to plasma-membrane H<Superscript>+</Superscript> flows and comprise separate photo- and dark-operated pathways

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H⁺ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H⁺ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1–2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (Q_A) via two separate (photochemical and non-photochemical) pathways.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. II. Alternative spectral forms and inhibitor effects on kinetics of Signal II in flashing light

Linewidth and hyperfine structure measurements of the EPR spectrum of Signal II in spinach chloroplasts show that the signal reflects two alternative states. One state is characterized by a 16-G linewidth and four partially resolved hyperfine components. The other state has 19 G linewidth and five partially resolved hyperfine components. It is possible to interconvert these two states by changing the ionic strength of the chloroplast suspension. Both states of Signal II show similar light-induced increases in dark-adapted chloroplasts and respond to 10-μs white light flashes with identical kinetics.

In chloroplasts at room temperature, Signal II dark decays to 50% of its total light-induced level in about 1 h. Single flashes increase the spin concentration in these aged chloroplasts but with decreased effectiveness compared with fresh, dark-adapted chloroplasts. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) decreases the decay time of Signal II from hours to seconds without appreciably altering the level of Signal II formed in saturating continuous light. However, both the formation time constant and the extent of Signal II increase stimulated by a single saturating flash are decreased in CCCP-treated chloroplasts.

These results are interpreted in terms of the model, proposed in the preceding paper, in which Signal II is generated by oxidation-reduction reactions on the water side of Photosystem II.