Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

New results on the properties of photosystem II centers blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea in their different photoactive states

We have studied the 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) action on the different S states by oxygen, fluorescence and luminescence measurements.We show that no oxygen is evolved during a flash following the addition of DCMU to centers in their S₃ state. This suggests that oxygen inhibition cannot be attributed solely to a blocking between Q and A. For all the photoinactive states, the only remaining pathway for the quencher reoxidation, in the presence of DCMU, appears to proceed through a back reaction. Therefore, the complete quencher regeneration still occurring when the fourth positive charge is formed in the presence of DCMU is also an indication of an action by DCMU at the donor side.The data well fit the model in which the oscillations of the fluorescence yield and their damping are attributed to a fast equilibrium between two forms of the centers: a photoactive and a photoinactive form, both of which are quenchers. The equilibrium constant depends on the number of positive charges stored and DCMU changes the characteristics of this equilibrium.     

The effects of carbon dioxide concentration on oxygen evolution and fluorescence transients in synchronous cultures of Chlorella pyrenoidosa

1. 1. The steady-state fluorescence yield of Chlorella pyrenoidosa is strongly affected by CO₂ concentration: the yield is approximately 2-fold higher in the presence than in the absence of CO₂. During induction, in the presence of saturating CO₂, accelerating oxygen evolution is paralleled by rising fluorescence (M₂-P₃ transient); in the absence of CO₂, fluorescence yield remains at the low M₂ level.

2. 2. Both illumination and CO₂ content are important in determining the steady-state fluorescence yield: at lower illuminations, lower concentrations of CO₂ are required to obtain a maximum fluorescence yield.

3. 3. The slow fluorescence transients are not affected directly by pH but only indirectly through the CO₂ concentration.

4. 4. The CO₂-dependent fluorescence rise (M₂-P₃ transient) is most readily observed in cells harvested early in the light period of a synchronous culture, but it can also be elicited in cells harvested during the dark period.

5. 5. Addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) to CO₂-deprived cells raises the fluorescence yield approximately 4-fold, that is to the same high level as cells supplied with CO₂ and DCMU.

6. 6. The effects of CO₂ provide a new example of a marked parallelism between photosynthetic electron transport and fluorescence. To explain such parallelism, it seems necessary to postulate large changes in the de-excitation processes within Photosystem II units or in the distribution of excitation between Photosystems I and II.

Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening)

Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) Inhibition of System II and Light-Induced Regulatory Changes in Energy Transfer Efficiency

Oxygen pulses produced in Chlorella by a xenon flash of 15 μsec half-width were measured by means of a rapid oxygen polarograph. Under appropriate conditions the height of the pulse caused by a saturating flash was a measure of the number of active reaction centers in system II. In pigment state II, caused by illumination during several minutes with light II, the number of active centers II was the same as in pigment state I. Oxygen pulses produced by about half-saturating flashes were diminished by about 7-10% in state II, showing that the fluorescence decrease in light II was at least partly caused by a decrease in energy transfer to reaction center II. After addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), only the first flash produced oxygen which gives additional support for the hypothesis that DCMU inhibits between Q and system I.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

UV-B radiation-induced donor- and acceptor-side modifications of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.     

Dichlorophenylurea-resistant oxygen evolution in Chlorella after cerulenin treatment

Fluorescence spectra at 77 K, oxygen evolution at 30°C and delayed fluorescence at 25°C were measured in Chlorella pyrenoidosa cultures with and without cerulenin and subsequent 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment, respectively. In pure algal cultures the oxygen evolution was inhibited by DCMU and the long-time component of fluorescence was, highly influenced by DCMU, as expected. In contrast, both oxygen evolution and delayed fluorescence became DCMU-resistant in cerulenin-treated cultures. The DCMU-resistance is correlated with a change in the fatty acid distribution of the thylakoid membrane, which also leads to changes in the prompt fluorescence. Cerulenin appears to be a promising new tool of diagnostics for the hitherto unsatisfactorily understood processes of oxygen evolution in photosynthesizing organisms.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Comparison of ATP-induced and DCMU-induced increases of chlorophyll fluorescence

A comparative study of the ATP-induced and the DCMU-induced increases of dark chlorophyll fluorescence after activation of the latent ATPase gave the following results: (1) The ATP-induced fluorescence rise exceeds the DCMU-induced rise by an amount equivalent to the rapid component of the biphasic ATP-induced change. There is complementarity between the slow component and any preceding DCMU-induced fluorescence rise. (2) Up to 10^?4 M DCMU (3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea)), with the slow component being completely suppressed, the rapid ATP-induced phase is unaffected. It becomes eliminated, though, with an I₅₀ of about 3 · 10^?4 M. (3) No binary oscillations in dependence of the number of preilluminating flashes are observed for the rapid ATP-induced fluorescence increase. Under identical conditions such oscillations are found upon DCMU-addition. (4) The amplitude of the rapid ATP-induced fluorescence rise is unaffected by closure of Photosystem II reaction centers in presence of DCMU and NH₂OH by a single saturating flash (removal of about 50% of total quenching). With further flashes and gradual complete removal of quenching, the rapid ATP-induced change is eliminated with a two-step dependency. It is concluded that the rapid phase of the ATP-induced increase in fluorescence reflects reverse electron flow at non-B-type reaction centers, while the slow phase is linked to reverse electron flow at B type centers. On the basis of these results a model is proposed for heterogeneous interactions between the ATPase and B-type and non-B-type electron-transport chains. ‘Direct coupling’ appears to be possible between CF₀-CF₁ and those electron-transport chains which are located in the stroma-exposed margin region of the grana stacks (PS IIβ units with non-B-type properties).     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

The kinetic relationship between the C-550 absorbance change,the reduction of Q(ΔA320) and the variable fluorescence yield change in chloroplasts at room temperature

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolated, envelopefree chloroplasts in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isosbestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differential extinction coefficient Δ?(540–550) is calculated to be approximately 5 mM^?1 · cm^?1. The kinetics of the C-550 absorbance change, occurring upon the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical conditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The light-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers.It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Thus, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at room temperature.     

Characterization of photosystem ii electron acceptors in Phormidium laminosum

Chlorophyll a fluorescence has been used to monitor the redox state of the primary electron acceptor of photosystem II (PS II) in the blue-green alga Phormidium laminosum during equilibrium titrations. The shape of induction curves measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) have been analyzed. The induction curves were very similar in unfractionated thylakoid membranes and PS II particles. In both, the fast (alpha) phase was sigmoidal, and was followed by a slow (beta) exponential tail. Thus, the structural organization and complexity of the particles (J. M. Bowes and P. Horton, Biochim, Biophys. Acta 680, 127-133 (1982), as indicated by the occurrence of energy transfer between alpha centers and presence of beta centers, must preexist in the membranes. Redox titration of the initial level of fluorescence indicated the presence of a single quencher QH in the unfractionated thylakoids, midpoint potential: Em7.0 approximately -35 mV (n = 1). Thus, the occurrence of a single acceptor is characteristic for P. laminosum and the absence of a low potential acceptor in PS II particles (J.M. Bowes, P. Horton, and D.S. Bendall, FEBS Lett. 135, 261-264 (1981] was not the result of its removal during their preparation. The midpoint potential of Q varied by -60 mV/pH unit in PS II particles and membrane fragments, with a pK at pH greater than 8.5 (particles) and at pH 7.5 (fragments). In PS II particles, DCMU raised the pK by approximately 0.5 pH units. It is argued that the pH dependence of Q is conferred by protonation of a protein which accompanies its reduction rather than protonation of the semiquinone Q X itself.     

Slow fluorescence quenching of Type A chloroplasts. Resolution into two components

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (> 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts.Ionophore-reversible quenching is identified with the Mg²⁺-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301–313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., and Zimmermann, U., eds.), pp. 281–288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264–274).The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

Energy transfer and quantum yield in Photosystem II

The photoreduction and dark reoxidation of Q_α and Q_β, the primary electron acceptors of Photosystems (PS) IIα and IIβ, respectively, in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) were studied in tobacco chloroplasts by means of fluorescence and absorbance measurements. The magnitude of a correction for an absorbance change by the oxidizing side of PS II needed in our previous study of the quantum yield of Q reduction (Biochim. Biophys. Acta 635 (1981), 111–120) has been determined. The absorbance change occurs in PS IIα mainly. The maximum fluorescence yield was found to be the same as in the mutant Su/su, which has a 3-fold higher reaction center concentration and a lower PS IIα to PS IIβ ratio. The kinetics of the light-induced fluorescence increase were measured after various pretreatments and the corresponding kinetics of the integrated fluorescence deficit were analyzed into their α and β components. From the results the contribution to the minimum fluorescence level, the degree of energy transfer between units, and the quantum efficiency of Q reduction were calculated for both types of PS II. This led to the following conclusions. The absence of energy between PS IIβ antennae is confirmed. Fluorescence quenching in PS IIα was adequately described by the matrix model, except for a decrease in the energy transfer between units during photoreduction of Q_α, possibly due to the formation of ‘islets’ of closed centers. PS II reaction centers in which Q is reduced do not significantly quench fluorescence. The ratio of variable to maximum fluorescence, 0.77 in PS IIα and 0.92 in PS IIβ, multiplied by the fraction of Q remaining in the reduced state after one saturating flash, 0.88 in PS IIα and greater than 0.95 in PS IIβ, leads to a net quantum efficiency of Q reduction in the presence of DCMU and NH₂OH of 0.68 in PS IIα and about 0.90 in PS IIβ. These values are in good agreement with the measured overall quantum efficiency of Q reduction.     

Functional analysis of photosystem II in a PsbO-1-deficient mutant in Arabidopsis thaliana

The Arabidopsis thaliana mutant psbo1 (formerly the mutant LE18-30), which contains a point mutation in the psbO-1 gene leading to defective expression of the PsbO-1 protein, has recently been described [Murakami, R. et al. (2002) FEBS Lett. 523, 138-142]. This mutant completely lacks the PsbO-1 protein and overexpresses the PsbO-2 protein. To further study the effect of PsbO-1 deficiency on the function of photosystem II, the polyphasic chlorophyll a fluorescence rise and flash fluorescence induction and decay of the relative fluorescence quantum yield were measured in whole leaves from wild type and the psbo1 mutant. Additionally, flash oxygen yield experiments were performed on thylakoid membranes isolated from wild type and the psbo1 mutant. The results obtained indicate that during fluorescence induction the psbo1 gene exhibited an enhanced O to P transition. Additionally, while the J to I transition in wild type accounted for more than 30% of the total fluorescence yield, in the mutant it accounted for less than 2% rise in the total. Analysis of the flash-induced fluorescence rise in the presence of DCMU indicated that in wild type the ratio of PS IIalpha to PS IIbeta reaction centers was approximately 1.2 while in the mutant the ratio was approximately 0.3. Fluorescence decay kinetics in the absence of DCMU indicated that electron transfer to QB was significantly altered in the mutant. Fluorescence decay kinetics in the presence of DCMU indicated that the charge recombination between QA- and the S2 state of the oxygen-evolving complex was retarded. Furthermore, flash oxygen yield analysis indicated that both the S2 and S3 states exhibited significantly longer lifetimes in the psbo1 mutant than in wild type. Our data indicate that while PsbO-1-deficient plants can grow photoautotrophically (although at a reduced growth rate) the photochemistry of PS II is significantly altered.     

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.