Catalytic Site Of Water Oxidation Research Articles

Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy.

Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

Dynamic pH-induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen.

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β-barrel domain in response to the fluctuating acidity of the thylakoid lumen.

FTIR spectroelectrochemistry combined with a light-induced difference technique: Application to the iron-quinone electron acceptor in photosystem II

Photosystem II (PSII) in plants and cyanobacteria performs light-driven water oxidation to obtain electrons necessary for CO2 fixation. In PSII, a series of electron transfer reactions take place from the Mn4CaO5 cluster, the catalytic site of water oxidation, to a plastoquinone molecule via several redox cofactors. Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been extensively used to investigate the structures and reactions of the redox cofactors in PSII. Recently, FTIR spectroelectrochemistry combined with the light-induced difference technique was applied to study the mechanism of electrontransfer regulation in PSII involving the quinone electron acceptors, QA and QB, and the non-heme iron that bridges them. In this mini-review, this combined FTIR method is introduced, and obtained results about the redox reactions of the non-heme iron and QB, involving the long-range interaction of the Mn4CaO5 cluster with the electron-acceptor side, are summarized.

No Evidence from FTIR Difference Spectroscopy That Aspartate-170 of the D1 Polypeptide Ligates a Manganese Ion That Undergoes Oxidation during the S0 to S1, S1 to S2, or S2 to S3 Transitions in Photosystem II

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.

Evaluation of different mechanistic proposals for water oxidation in photosynthesis on the basis of Mn4OxCa structures for the catalytic site and spectroscopic data

Recent progress in EPR and EXAFS spectroscopy, quantum mechanical calculations and X-ray crystallography led to a tremendous improvement in our picture of the catalytic site of water oxidation in photosystem II. It is now likely that the four Mn ions are grouped in a 3 + 1 fashion with three short Mn–Mn distances of about 2.7 A and one long Mn–Mn distance of 3.3 A. In addition, Ca has been firmly localized close to the Mn centers, with an average distance of 3.4 A and an average angle of the Mn–Ca vectors close to the membrane normal (≤23°). The recent crystal structure of Ferreira et al. (Science, 2004, 303, 1831–1838) suggests that the Mn3 unit and Ca form a distorted cubane like structure. The fourth Mn is ‘dangling’ from this unit either via a μ4-oxo bridge or a mono μ-oxo bridge. However, the precise Mn–Mn distances and the bridging situation still need to be worked out. On this structural basis and the available spectroscopic data two possible mechanisms for photosynthetic water oxidation are discussed in order to highlight questions that still need to be solved for a full understanding of this fascinating reaction.

A two-electron reaction at the catalytic site of water oxidation in manganese-depleted Photosystem II vesicles in the presence of an electron donor. Coupled electron and proton transfer processes

We have studied the electron and proton transfer at the manganese-depleted catalytic site of water oxidation in the presence of the electron donor 1,5-diphenylcarbazide (DPC). On the basis of measurements of flash-induced fluorescence yield, electrochromic bandshifts and pH transients into the aqueous medium, we have been led to the following conclusions: (1) The electron donor DPC was involved in a two sequential photostep process just like Mn 2+ in photoactivation. After a first flash, the product of the reaction of DPC with the first Photosystem II charge separation was unstable (half-decay around 1 s at pH 6.5) until converted into a stable one by a second flash. The formation of the unstable intermediate was rather favored at alkaline pH. Its concentration increased progressively with pH to reach a maximum at pH 7.2–7.4. Nevertheless, consumption of this intermediate required a proton binding, since its regeneration after the second flash needed a latency dark time inversely proportional to proton concentration. (2) Each step of the process was accompanied by proton exchange with the medium. At acidic pH, the odd flashes in a series induced acidification, whereas on even flashes occurred alkalinization. The pattern of proton release/uptake varied extremely as a function of pH, between 4 H + and −2 H +/e −, with opposite changes at acidic and basic pH. At neutral pH, 1 H +/e − was assumed to be released at each step. On the basis of these results and data already in the literature, the molecular details of the pH-dependent part of the deprotonation are discussed in terms of electrostatic interactions between the polarized catalytic center and bound H + and OH − ions. The described two-photostep process is proposed to reflect the two-electron water oxidation to an hydrogen peroxide intermediate.

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Functionally important domains of the large hydrophilic loop of CP47 as probed by oligonucleotide-directed mutagenesis in Synechocystis sp. PCC 6803

The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which roughly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in delta 1 (I265-F268), delta 2 (T271-K277), delta 4 (T304-L309), delta 5 (F311-N317), and delta 12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains delta 3 (K277-E283) and delta 11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain delta 8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains delta 6 (G333-I336), delta 7 (K347-R352), delta 9 (V392-Q394), and delta 10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (delta 6, delta 7, delta 8, delta 9, delta 10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.

The manganese stabilising protein (MSP) of Prochlorothrix hollandica is a hydrophobic membrane-bound protein

The oxygen-evolving Photosystem II complex of the chlorophyll-a b- containing prokaryote Prochlorothrix hollandica is similar to that of cyanobacteria in terms of the absence of the 23 kDa extrinsic polypeptide. In contrast to cyanobacteria and green chloroplasts, the manganese stabilizing protein (MSP) of Prochlorothrix is a hydrophobic protein as detected by Triton X-114 phase partitioning and has an apparent molecular mass of 37 kDa. The catalytic site of water oxidation in Prochlorothrix thylakoids is significantly more resistant to inhibition by hydroxylamine as compared with thylakoids of Chlamydomonas or Synechocystis 6803. These results indicate that the properties of the donor side of Photosystem II complex of Prochlorothrix differ from that of the related cyanobacteria.

Mass spectroscopic analysis of N 2 formation by flash-induced oxidation of hydrazine and hydroxylamine in normal and tris-treated tobacco chloroplasts

The flash-induced dinitrogen evolution was analyzed in normal and Tris-inhibited tobacco chloroplasts ( Nicotiana tabacum var. John William's Broadleaf) which were dark-incubated (at least 20 min) in the presence of NH 2NH 2 and/or NH 2OH. (a) The N 2 yield due to a single turnover flash as a function of NH 2NH 2 concentration is practically the same in both normal and Tris-washed chloroplasts, even in a range where the oxygen evolution remains almost unaffected by NH 2NH 2. Illumination with ten flashes leads to higher integral N 2 evolution in Tris-washed samples at limiting NH 2NH 2 concentrations. (b) Under aerobic conditions oxidation of NH 2NH 2 and NH 2OH is coupled with significant O 2 uptake. Both N 2 formation and O 2 uptake remain unaffected by DCMU in a single turnover flash but become drastically inhibited under multiple flash excitation. (c) The N 2 yield per flash due to PS-II-induced NH 2NH 2 oxidation markedly depends on the O 2 content of the suspension. This effect is also observed in the presence of superoxide dismutase (SOD). (d) If chloroplasts are incubated with an equimolar ratio of 14NH 2 14NH 2 and 15NH 2 15NH 2, hardly any 14N 15N formation is detected, indicating that the nitrogen-nitrogen bond remains intact during the oxidation process. A totally different isotope distribution is obtained in the case of an equimolar mixture of 14NH 2OH and 15NH 2OH which reflects a statistical bimolecular reaction between NHOH radicals. In a mixture of 14NH 2OH and 15NH 2 15NH 2 the ratio of mass 29 to mass 30 increases with increasing 14NH 2OH content. Based on the present results, flash-induced N 2 formation is inferred to occur via univalent oxidation of NH 2OH and NH 2NH 2 either by P-680 + or by the functionally connected tyrosine(s) of polypeptide D-1 (Y ox Z) and/or D-2 (Y ox D). NH 2OH and NH 2NH 2 do not interact with the S 1 state of the catalytic site of water oxidation in a way that leads to flash-induced N 2 production.

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680(+) was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680(+) reduction by Y z under conditions where the catalytic site of water oxidation stays in the redox state S1. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca(2+). The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the QB-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680(+) reduction by Y z , and (c) Ca(2+) could play a functional role for the electronic and vibrational coupling between the redox groups Y z and P680. The electron transport from Y z to P680(+) is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.

Temperature dependence of P680 + reduction in O 2-evolving PS II membrane fragments at different redox states S i of the water oxidizing system

The electron transfer kinetics from Z to P680 + was analyzed as a function of temperature in the range of 248< T< 295 K by measuring absorption changes induced at 830 nm by a laser flash train in dark adapted O 2 evolving PS II membrane fragments from spinach. It was found: (i) that the kinetics of P680 + reduction and their dependence on the redox state S i of the catalytic site of water oxidation are only slightly affected by temperature within the physiological range of 270< T<295 K. (ii) In the dark relaxed state S, the electron transfer from Z to P680 + exhibits an activation energy of the order of 10 kJ/mol in 248< T<295 K. (iii) In the 2nd and subsequent flashes of the train the ability for a stable charge separation between P680 + and Q − A, markedly decreases below −10°C. This phenomenon is assumed to be due to a strong effect of temperature on the electron transfer from Q − A to Q B. The results are briefly discussed in relation to possible effects of structural changes in the D-1/D-2 polypeptide complex on the reaction coordinate of electron transfer steps in PS II.