Water-oxidizing Complex Research Articles

Assignment of histidine residues in Dl protein as possible ligands for functional manganese in photosynthetic water-oxidizing complex

By use of a chemical modifier, the ligand amino-acid residue(s) for manganese ligation in the photosynthetic water-oxidation center was investigated. The following was found. (1) Treatment with diethyl pyrocarbonate (DEPC), a histidine modifier, caused a loss of photoactivation capability of NH2OH-treated PS II membranes (devoid of Mn). (2) DEPC-induced loss of photoactivation capability showed pH dependence with a pKaof 6.9 and was reversed by subsequent treatment with NH2OH, both indicative of specific involvement of histidine residue in the modification. (3) DEPC modification was protected by the presence of Mn photoligated in the center, and DEPC-modified centers drastically lost the Mn binding affinity accompanied by a decrease in rate of generation of the unstable intermediate state involved in the multi-quantum process of photoactivation. (4) [14C]DEPC treatment radiolabelled several PS II proteins, but the labelling intensities of D1 and a 60 kDa band due to D1/D2 heterodimer were specifically suppressed among others by the presence of photoligated Mn. Based on this, it was inferred that D1 protein contains a ligand histidine residue(s) specifically interacting with Mn functional in water oxidation.

The reaction of H 2S with the photosynthetic water-oxidizing complex and its lack of reaction with the primary electron acceptor in spinach

Inhibition of photosynthetic water oxidation by H 2S, a substrate analog, has been investigated using equilibrium titrations and EPR spectroscopic detection of the electron donors in spinach Photosystem II (PS II) membranes and compared to inhibition by NH 2OH. Like NH 2OH, H 2S inhibits formation of the S 2 oxidation state of the water oxidizing complex by a two-step process in the dark. Initially, reversible inhibition of S 2 occurs upon binding to a high affinity site in the dark (S 1) state at a low concentration of inhibitor (50% inhibition: 0.07 μmol H 2S/mg Chl, corresponding to about 15 H 2S/PS II). This causes no loss of steady-state O 2 evolution and can be reversed by illumination at room temperature, which causes multiple turnovers. At higher concentrations, irreversible inhibition occurs due to the cooperative release of 3 out of 4 Mn 2+/PS II using mild washing conditions, with parallel loss of O 2 evolution activity. This stoichiometry of Mn release is preserved throughout the entire concentration range of inhibition by both H 2S and NH 2OH, suggesting a common binding site for at least 3, and possibly all 4, of the Mn ions which are present in PS II. This consistent with independent earlier work showing the net release of 4 Mn/PS II using stronger washing conditions, and also with EPR spectroscopic evidence assigning the S 2 multiline signal to a cluster of 3–4 Mn ions. The concentration of H 2S which induces 50% irreversible inhibition is 17-fold greater than that required for NH 2OH. Qualitatively, the weakerinhibition by H 2S is consistent with its lower oxidation potential compared to N 2OH. However, the quantitative agreement is poor, suggesting that other environmental factors must be involved in determining their relative inhibition strenghts. Unlike NH 2OH, H 2S does not affect the structure of the primary quinone electron acceptor, Q A −-His-Fe (structure by analogy to bacterial reaction centers), as seen by formation of the normal EPR signals at g = 1.8 and g = 1.9 for the photoreduced semiquinone, instead of shifted resonance at g = 2.1 observed with NH 2OH. H 2S is therfore unable to bind to the NH 2OH site on the acceptor side of PS II.

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii.

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.

Inhibition of electron transport in photosystem II by hydroxylamine: further evidence for two binding sites

The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity to a site that structurally interacts with the primary electron acceptor FeQ/sub A//sup -/. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQ/sub A/) to g = 2.1 on the first turnover. These results indicate that the binding site for NH/sub 2/OH overlaps with or interacts with the binding site for Q/sub B/. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH/sub 2/OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH/sub 2/OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, whichmore » exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. The authors also find that the high-affinity binding of NH/sub 2/OH within the WOC produces no observable EPR-active products in the dark. They conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b/sub 559/ or signal II (/sup 160/Tyr x D/sub 1/ protein).« less

Binding of hydroxylamine to the water-oxidizing complex and the ferroquinone electron acceptor of spinach photosystem II

Binding of hydroxylamine to the water-oxidizing complex and the ferroquinone electron acceptor of spinach photosystem II

Mn2+/Mn3+ and Mn3+/Mn4+ mixed valence binuclear manganese complexes of biological interest

Binuclear and multinuclear manganese containing enzymes which occur in biology are involved in functions such as hydrogen peroxide decomposition in bacteria and the oxidation of water to oxygen during photosynthesis, respectively. In the case of the water-oxidizing complex, the so-called S/sub 2/ oxidation state has been characterized by EPR spectroscopy to be a mixed valence manganese cluster. This can be produced in two forms. The native form, which is found in active O/sub 2/-evolving samples, is considered to be either tetranuclear or trinuclear in Mn, based upon interpretation of its broad 1500 G-wide 19-line multiline EPR signal and unusual temperature dependence. A partially decoupled form is found in inactivated samples having a narrow width (1345 G), with only 16 lines and a more Curie-like temperature dependence, all features which are found in typical Mn/sup 3 +//M/sup 4 +/ binuclear centers (16-line EPR, spin S = 1/2 ground state). For the native form both different oxidation states and different numbers for the manganese ions in the cluster have been proposed in order to account for the larger spectral width due to the hyperfine interaction with /sup 55/Mn. Both binuclear Mn/sup 2 +//Mn/sup 3 +/ and tetranuclear/sup 4/ 3Mn/sup 3 +//Mn/sup 4more » +/ states have been considered. Notably lacking in this analysis has been EPR data for binuclear complexes in which both types of mixed valence oxidation states, Mn/sup 3 +//Mn/sup 4 +/ and Mn/sup 2 +//Mn/sup 3 +/, are compared within the same ligand system. The present report gives the first experimental evidence showing how /sup 55/Mn hyperfine data can easily distinguish between binuclear manganese complexes having these mixed valence oxidation states.« less

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.

Photoactivation of the water-oxidizing complex in Photosystem II membranes depleted of Mn and extrinsic proteins. I. Biochemical and kinetic characterization

Conditions were defined for obtaining photoligation of Mn 2+ and photoactivation of O 2 evolution in NH 2OH- and Tris-extracted PS II membranes (TMF-2) completely devoid of the CF 0/CF 1 complex and containing only approx. 2–3 PS II e − acceptor equivalents per PS II reaction center. At optiomal pH (pH 6.5) only light and Mn 2+ were essential for Mn ligation by the apo-S-state complex (approx. 4 Mn/reaction center); however, Ca 2+ addition was required for maximal expression of water oxidation activity of the photoligated Mn. In the absence of added PS II e − acceptors, the quantum efficiency and yield of photoactivation was diminished and was insensitive to atrazine. PS II e − acceptors increased the quantum efficiency/yield by approx. 2-fold and conferred sensitivity to atrazine. Kinetic analyses of the photoactivation process gave evidence for a rate constraint ( t 1 2 ≈ 125 ms ) and an unstable intermediate (half-life of approx. 1.0 s). Cl − was not absolutely essential for photoactivation. A 7-fold increase of rate of O 2 evolution was obtained without Cl − addition to Cl −-depleted NH 2OH-TMF-2; Cl − addition during photoactivation gave only a 2-fold additional increase. This Cl − effect ( K m = 3.8 mM) was different from the Cl effect ( K m = 1.5 mM) on O 2 evolution. Weak light ageing of NH 2OH-TMF-2 in the absence (but not in the presence) of Mn 2+ inhibited photoactivation and photoreduction of DCIP by Mn 2+; however, DCIP photoreduction by DPC or TPB was not diminished. Such weak light ageing also increased the VLP-form of Cyt b-559, but there was no apparent correlation between the increase in O 2 evolution and the conversion of the VLP- and LP-Cyt b-559 to the HP-Cyt b-559 during photoactivation. Photoactivation is suggested to be a two-quantum process in which two Mn 2+ are sequentially bound, photooxidized and ligated by the apo-S-state complex. This process facilitates ligation of two additional Mn ions to form the tetra-Mn S-state water-oxidizing complex.

Evidence for an association between a 33 kDa extrinsic membrane protein, manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S1 → S2 reaction, as seen by the multiline EPR signal for the S2 state of the water-oxidizing complex, while reversibly inhibiting O2 evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O2 evolution and the S2 multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl2 to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O2 evolution and S2 multiline yield. These depleted membranes do not oxidize water or produce the normal S2 state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.

The Water Oxidation Complex of Chlamydomonas

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl(2) washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O(2) evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.

Manganese-histidine cluster as the functional center of the water oxidation complex in photosynthesis

The recent model of Kambara and Govindjee for water oxidation [Kambara T. and Govindjee (1985) Proc. Natl. Acad. Sci. U.S.A., 82:6119-6123] has been extended in this paper by examining all the data in order to identify the most likely candidate for the 'redox-active ligand' (RAL), suggested to operate between the water oxidizing complex (WOC) and Z, the electron donor to the reaction center P680. We have concluded that a very suitable candidate for RAL is the imidazole moiety of a histidine residue. The electrochemical data available on imidazole derivatives play heavily in this identification of RAL. Thus, we suggest that histidine might play the role of an electron mediator between the WOC and Z. A model of S-states in terms of their plausible chemical identity is presented here.

On the action of hydroxylamine, hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S0 → S1, S1 → S2, S2 → S3, S3 → (S4) → S0. State S1 is most stable in the dark. Oxygen is released during S3 → (S4) → S0. Hydroxylamine and hydrazine interact with S1. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S1 → S2 : S2 → S3 : S3 → (S4) → S0 : S0 → S1 :... in the absence of the agents, to S1 (*) → S0 : S0 → S1 : S1 → S2 : S2 → S3 :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH(+)(S1 → S2) : 1H(+)(S2 → S3) : 2H(S3 → S0) : 1H(+)(S0 → S1) :... to 2H(+)(S1 (*) → S0) : 1H(+)(S0 → S1) : OH(+)(S1 → S2) : 1H(+)(S2 → S3) : 2H(+)(S3 → S0) :... The 2H(+) were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53-57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n≥3 molecules of hydroxylamine and of n≥2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl(-) or OH(-), which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S1.

Electrochemistry and stability studies of oxo-bridged dinuclear ruthenium(III) complexes for water oxidation

Electrochemical and water oxidation studies have been carried out for the bridged complexes (1) and (2). [graphic omitted] Three consecutive reversible two-electron processes were shown to occur. Repeated continuous cyclic scans of the redox processes, however, led to the decomposition of the dimers to the monomeric complex. Visible spectra showed that the decomposition leads to the formation of cis-[Ru(bpy)–2(H2O)2]2+. The dimer complexes are stable at a Nifion-coated graphite electrode in their adsorbed state even on repeated cyclic scans of the redox processes. It was shown by gas chromatography as well as by mass spectroscopy that oxygen was evolved by water oxidation on adding CeIV ion to an aqueous solution of the dinuclear complex. The turnover number showed that the complexes worked as catalysts for water oxidation, although they suffer partial decomposition during the catalysis. The nitrite complex was more stable than the water complex against decomposition.

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II.

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.

Cooperative and reversible action of three or four hydroxylamine molecules on the water-oxidizing complex

Photosynthetic water oxidation is a four-step oxidation process which is formally described by the transitions S 0 → S 1, S 1 → S 2, S 2 → S 3, S 3 → S 4 → S 0. In dark-adapted thylakoids S 1 is the most stable and oxygen is liberated after abstraction of three oxidizing equivalents from the water-oxidizing complex. If hydroxylamine is present at low concentrations (15–100 μM), five instead of three oxidizing equivalents have to be abstracted before dioxygen is liberated. We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. We obtained an apparent Hill coefficient of 2.43. This result could be explained by cooperative binding of 3 or 4 hydroxylamine molecules, and by the assumption that each center which was modified by >= 1 hydroxylamine molecule gave rise to the same modified pattern of proton release.

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 NH 2OH (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 NH 2OH or NH 2NH 2 are uninhibited by Cl − depletion. Addition of NH 2OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH 2OH 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 S 2, but not to inhibit the transitions from S 0 to S 1, or from S 1 to S 2.