Kinetics of manganese redox transitions in the oxygen-evolving apparatus of photosynthesis

The kinetics of the S-state transitions of the oxygen-evolving complex were analyzed in dark-adapted, oxygen-evolving Photosystem-II preparations supplied with the electron acceptor 2,5-dichloro-p-benzoquinone. The kinetics of flash-induced absorbance changes at 350 nm, largely due to the successive S-state transitions S₀ → S₁, S₁ → S₂, S₂ → S₃ and S₃ →; S₀, confirm the +1, +1, +1, ?3 sequence of manganese oxidation reported earlier (Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9), and reveal half-times of 30, 110, 350 and 1300 μs, respectively, for these transitions.     

Absorbance difference spectra of the S-state transitions in Photosystem II core particles

Redox changes of the oxygen evolving complex in PS II core particles were investigated by absorbance difference spectroscopy in the UV-region. The oscillation of the absorbance changes induced by a series of saturating flashes could not be explained by the minimal Kok model (Kok et al. 1970) consisting of a 4-step redox cycle, S₀ S₁ S₂ S₃ S₀, although the values of most of the relevant parameters had been determined experimentally. Additional assumptions which allow a consistent fit of all data are a slow equilibration of the S₃ state with an inactive state, perhaps related to Ca²⁺-release, and a low quantum efficiency for the first turnover after dark-adaptation. Difference spectra of the successive S-state transitions were determined. At wavelengths above 370 nm, they were very different due to the different contribution of a Chl bandshift in each spectrum. At shorter wavelengths, the S₁ S₂ transition showed a difference spectrum similar to that reported by Dekker et al. 1984b and attributed to an Mn(III) to Mn(IV) oxidation. The spectrum of absorbance changes associated with the S₂ S₃ transition was similar to that reported by Lavergne 1991 for PS II membranes. The S₀ S₁ transition was associated with a smaller but still substantial absorbance increase in the UV. Differences with the spectra reported by Lavergne 1991 are attributed to electrostatic effects on electron transfer at the acceptor side associated with the S-state dependence of proton release in PS II membranes.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S₀ to S₄ redox state of the oxygen evolving complex - Z secondary electron donor of PS II     

Manganese K-edge X-ray absorption spectra of the cyclic S-states in the photosynthetic oxygen-evolving system

A set of Mn K-edge XANES spectra due to the redox states S₀–S₃ of the OEC were determined by constructing a highly-sensitive X-ray detection system for use with physiologically native PS II membranes capable of cycling under a series of saturating laser-flashes. The spectra showed almost parallel upshifts with relatively high K-edge half-height energies given by 6550.9±0.2 eV, 6551.7±0.2 eV, 6552.5±0.2 eV and 6553.6±0.2 eV for the S₀, S₁, S₂ and S₃ states, respectively. The successive difference spectra between S₀ and S₁, S₁ and S₂, and S₂ and S₃ states were found to exhibit a similar peak around 6552–6553 eV, indicating that one Mn(III) ion or its direct ligand is univalently oxidized upon each individual S-state transition from S₀ to S₃. The present data, together with other observations of EPR and pre-edge XANES spectroscopy, suggest that the oxidation state of the Mn cluster undergoes a periodic change; S₀: Mn(III,III,III,IV) S₁: Mn(III,IV,III,IV) S₂: Mn(III,IV,IV,IV) S₃: Mn(IV,IV,IV,IV) or Mn(III,IV,IV,IV)·L⁺ with L being a direct ligand of a Mn(III) ion.Abbreviations Chl chlorophyll - D tyrosine 160 on the D2 protein, an accessory electron donor in PS II - D⁺ the oxidized form of D - EDTA ethylene-diaminetetraacetic acid - EPR electron paramagnetic resonance - EXAFS extended X-ray absorption fine structure - HL py-2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol - Mes 2-(N-morpholino)ethanesulfonic acid - N4 py-tris(2-pyridylmethyl)amine - OEC oxygen evolving complex - P680 primary electron donor of PS II - PS II Photosystem II - Q₄₀₀ a high spin Fe³⁺ of the iron-quinone acceptor complex in PS II - SSD solid state detector - XAFS X-ray absorption fine structure - XANES X-ray absorption near edge structure     

Inhibition of Photosystem II by formate. Possible evidence for a direct role of bicarbonate in photosynthetic oxygen evolution

In broken chloroplasts the presence of 100 mM sodium formate at pH 8.2 will specifically lengthen the Photosystem II relaxation times of the reactions S′₂ → S₃ and S′₃ → S₀. Rates of reactions S′₀ → S₁ and S′₁ → S₂ remain unaffected. Evidence is presented which indicates the discrimination among S-states by formate cannot be attributed to a block imposed on the reducing side of Photosystem II. The results are interpreted in context of the known interaction of formate and CO₂ which is bound to the Photosystem II reaction center complex. It is proposed that those S-state transitions which show extended relaxation times in the presence of formate must result in the momentary release and rebinding of CO₂. Furthermore since formate is acting on the oxygen-evolving side of Photosystem II, it would seem that CO₂ is released in reactions that occur there. A chemical model of oxygen evolution is presented. It is based on the hypothesis that hydrated CO₂ is the immediate source of photosynthetically evolved oxygen and explains why, under certain conditions formate slows only the S-state transitions S′₂ → S₃ and S′₃ → S₀.     

Optical characterization of Photosystem II electron donors

Detailed absorbance difference spectra are reported for the Photosystem II acceptor Q, the secondary donor Z, and the donor involved in photosynthetic oxygen evolution which we call M. The spectra of Z and Q could be resolved by analysis of flash-induced kinetics of prompt and delayed fluorescence, EPR signal II_f and absorbance changes in Tris-washed system II preparations in the presence of ferricyanide and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The spectrum of Z oxidation consists mainly of positive bands at 260, 300 and 390–450 nm on which a chlorophyll a band shift around 438 nm is superimposed, and is largely pH-independent as is also the case for the spectrum of Q reduction. The re-reduction of Z⁺ occurred in the millisecond time range, and could be explained by a competition between back reaction with Q^? (120 ms at pH 6.0) and reduction by ferrocyanide. When the Tris treatment is omitted the preparations evolve oxygen, and the photoreduction of Q (with DCMU present) is accompanied by the oxidation of M. The Q spectrum being known, the spectrum of the oxidation of M could be determined as well. It consists of a broad, asymmetric increase peaking near 305 nm and of a Chl a band shift, which is about the same as that accompanying Z in Tris-washed system II. Comparison with spectra of model compounds suggests that Z is a bound plastoquinol which is oxidized to the semiquinone cation and that the oxidation of M is an Mn(III) → Mn(IV) transition.     

Studies on the water-oxidizing system by the effects of different treatments in chloroplasts

Treatments such as trypsinization (50 μg/ml per mg Chl for 1 h), osmotic shock of the chloroplasts or mild heating altered the oxygen evolution in such a way that the properties of the Photosystem II were simplified. After these treatments, the damping of the oscillation pattern of O₂ yields induced by a flash series remained the same, irrespective of the level of inhibition induced by the treatment. This damping did not decrease with increasing flash energy, as observed in untreated chloroplasts. The light saturation curve of the S₂ → S₃ transition of the O₂ evolving system no more exhibited the slow-increasing phase at high flash energy observed under normal conditions. The kinetic properties of the O₂-evolving system were also simplified. After the treatments cited above, deactivation of S₂ and S₃ were identical and accelerated with respect to untreated chloroplasts. Turnover kinetics of the transitions S₁ → S₂ and S₂ → S₃ were also similar and simpler without a lag for S₂ → S₃. These results indicate that the treatments mentioned above disconnect one donor from the O₂-evolving complex. This donor, under normal conditions, contributes to the increase of the quantum yield of the transition S₂ → S₃ at high flash energy. This donor is here denoted by D. Our results are in agreement with the following working hypothesis: the large miss, observed on the S₂ → S₃ transition without any contribution of the donor D, may be due to the fact that the system needs a conformation change of the O₂-evolving complex in the S₂ state, so that the main donor Y can oxidize the second H₂O molecule in the water-splitting complex. In the inactive state corresponding to the absence of a conformation change, the donor D, being different in configuration, is likely to oxidize the S₂ state into an S₃ state at high light intensity.     

Flash-induced redox changes in oxygen-evolving spinach Photosystem II core particles

Flash-induced redox reactions in spinach PS II core particles were investigated with absorbance difference spectroscopy in the UV-region and EPR spectroscopy. In the absence of artificial electron acceptors, electron transport was limited to a single turnover. Addition of the electron acceptors DCBQ and ferricyanide restored the characteristic period-four oscillation in the UV absorbance associated with the S-state cycle, but not the period-two oscillation indicative of the alternating appearance and disappearance of a semiquinone at the Q_B-site. In contrast to PS II membranes, all active centers were in state S₁ after dark adaptation. The absorbance increase associated with the S-state transitions on the first two flashes, attributed to the Z⁺S₁ZS₂ and Z⁺S₂ZS₃ transitions, respectively, had half-times of 95 and 380 s, similar to those reported for PS II membrane fragments. The decrease due to the Z⁺S₃ZS₀ transition on the third flash had a half-time of 4.5 ms, as in salt-washed PS II membrane fragments. On the fourth flash a small, unresolved, increase of less than 3 s was observed, which might be due to the Z⁺S₀ZS₁ transition. The deactivation of the higher S-states was unusually fast and occurred within a few seconds and so was the oxidation of S₀ to S₁ in the dark, which had a half-time of 2–3 min. The same lifetime was found for tyrosine D⁺, which appeared to be formed within milliseconds after the first flash in about 10% inactive centers and after the third and later flashes by active centers in Z⁺S₃.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - D secondary electron donor of PS II - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S_0–3 redox state of the oxygen-evolving complex - Z secondary electron donor of PS II     

Structured near-infrared Magnetic Circular Dichroism spectra of the Mn4CaO5 cluster of PSII in T. vulcanus are dominated by Mn(IV) d-d ‘spin-flip’ transitions

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn₄CaO₅. VTVH MCD spectra taken in the S₂ state provide a clear g = 2, S = 1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749 nm, negative (?) at 773 nm and (+) at 808 nm are assigned as ⁴A → ²E spin-flips within the d³ configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S₃ exhibits a more intense (?) MCD peak at 764 nm and has a stronger MCD saturation characteristic. This S₃ MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S₂ EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.     

Electron transfer in the water-oxidizing complex of Photosystem II

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, –3 during the so-called S₀ S₁ S₂ S₃ S₀ redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S₂ and the lowest in state S₁.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.     

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn₄ cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S₂-, S₃-, and S₀-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.     

Flash-induced enhancements in the proton NMR relaxation rate of Photosystem II particles

Flash-induced enhancements in the NMR spin-lattice relaxation rate of solvent protons have been detected in suspensions of Photosystem II particles. The relaxation enhancements are small (less than 1% of background) and have been detected using signal-averaging techniques. The enhancements correlate with the known properties of the S states with respect to (1) decay kinetics, (2) extractants of manganese, (3) sensitivity to atrazine-type inhibition of electron transport, (4) reagents which accelerate S-state decay (ADRY reagents), and (5) the two-flash retardation in the S-state advancement that is produced by low concentrations of NH₂OH. The transient proton relaxation enhancement observed after a single flash arises from a strongly relaxing paramagnetic species that is produced by the S₁ → S₂ transition of the water-oxidizing center. The appearance of such a species on an oxidative transition is suggestive of an Mn(III) → Mn(IV) oxidation.     

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis — possible states and configurations of manganese and water

Absorption changes coupled with the individual transitions S₀–S₃ and redox reactions in the water-splitting enzyme system S of photosynthesis have been measured. The principal difficulties of measuring the very small absorption changes in the ultraviolet coupled with those reactions have been reduced drastically through the use of a highly purified Photosystem II complex isolated from the Cyanobacterium synechococcus. The general problem caused by the mixing of the S states during a train of flashes and the falsification through the overlap with absorption changes of Q_B (binary oscillations) have been treated as follows. (1) The binary oscillations were bypassed through the use of silicomolybdate and high concentrations of DCBQ, respectively, as external electron acceptor. (2) Stable absorption changes of the mixed S-state transitions have been deconvoluted through fitting procedures to get the changes of the individual transitions of S₁ → S₂ → S₃ → S₀ → S₁. (3) Kinetically resolved absorption changes of the S-states in the 100-μs range gave independent information on the individual transitions. (4) Stable absorption changes of the S₀ → S₁ transitions in the forefront were induced after shifting the S states through low concentrations of NH₂OH two units backwards. Analysis of the resulting sequence S_x → S₀ → S₁ → S₂ → S₃ → S₀, beginning with an NH₂OH depending pre-state, S_x, and followed by an S₀ → S₁ transition not mixed with the opposite S₃ → S₀ transition, increased the conclusiveness considerably. It results that the ultraviolet spectrum of the S₀ → S₁ transition is different from the spectra of the S₁ → S₂ and S₂ → S₃ transition. Possible states of manganese, water and surplus charges responsible for these spectra are presented.     

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Structural and catalytic properties of the oxygen-evolving complex. Correlation of polypeptide and manganese release with the behavior of Z+ in chloroplasts and a highly resolved preparation of the PS II complex

Treatment of intact thylakoid membranes with Triton X-100 at pH 6 produces a preparation of the PS II complex capable of high rates of O₂ evolution. The preparation contains four managanese, one cytochrome b-559, one Signal II_f and one Signal II_s per 250 chlorophylls. By selective manipulation of the preparation polypeptides of approximate molecular weights of 33, 23 and 17 kDa can be removed from the complex. Release of 23 and 17 kDa polypeptides does not release functional manganese. Under these conditions Z⁺ is not readily and directly accessible to an added donor (benzidine) and it appears as if at least some of the S-state transitions occur. Evidence is presented which indicates that benzidine does have increased access to the oxygen-evolving complex in these polypeptide depleted preparations. Conditions which release the 33 kDa species along with Mn and the 23 and 17 kDa polypeptides generate an alteration in the structure of the oxidizing side of PS II, which becomes freely accessible to benzidine. These findings are examined in relationship to alterations of normal S-state behavior (induced by polypeptide release) and a model is proposed for the organization of functional manganese and polypeptides involved in the oxygen-evolving reaction.