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20.2: The Kok Cycle and Oxygen Evolving Complex of Photosystem II

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    15047
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    Introduction

    We have just seen how photoexcitation of the non-reaction center chlorophyll turns that molecule into a good reducing agent, which transfers its electron to the nearest excited state level of the reaction center chlorophyll. If you count both steps together, the non-reaction center chlorophyll gets "photooxidized", in the process producing the "strong" oxidizing agent which is the positively charged chlorophyll derivative. The extra electron passed onto the second molecule will eventually be passed on to NADP+ to produce NADPH.

    These reactions occur in the presence of light and hence are called the light reactions. The light reactions of photosynthesis in green plants are shown in Figure \(\PageIndex{1}\) below, along with the standard reduction potentials of the participants, the Z scheme..

    photosynthSVGwithPPTX.svg
    Figure \(\PageIndex{1}\): Light reaction of photosynthesis and associated standard reduction potentials

    The combined processes of PSII and PSI resemble a "Z" scheme (rotate in your mind the standard reduction potential figure 90 degrees clockwise). In an organization reminiscent of electron transport in mitochondria, water is oxidized by photosystem II (PSII). Electrons from water are moved through PSII to a mobile, hydrophobic molecule, plastaquinone (PQ) to form its reduced form, PQH2. Another photosystem, photosystem I (PS1), is next in the electron transport pathway. It takes electrons from another reduced mobile carrier of electrons, plastocyanin (PCred) to ferredoxin, which becomes a strong reducing agent. Ferrodoxin is a protein with an Fe-S cluster (Fe-S-Fe-S in a 4 membered ring, with 2 additions Cys residues coordinating each Fe). It ultimately passes its electrons along to NADP+ to form NADPH. Note the complexes that produce a transmembrane proton gradient. In contrast to mitochondria, the lumen (as compared to the mitochondrial matrix) becomes more acidic than the stroma. Protons then can move down a concentration gradient through the C0C1ATPase to produce ATP required for reductive biosynthesis of glucose.

    Figure \(\PageIndex{2}\) below a more more detailed view of the molecular players in the light reaction.

    photosynth2.jpg
    Figure \(\PageIndex{2}\): Detailed View of Light Reaction of Photosynthesis (reprinted with permission from Kanehisa Laboratories and the KEGG project. Boxed number represent Enzyme Commission Number. Original KEGG Map with imbedded links.

    Photosystem II

    PSII has a complicated structure with many polypeptide chains, lots of chlorophylls, and Mn, Ca, and Fe ions. A Mn cluster, called the oxygen evolving complex, OEC (also called the OEX) is directly involved in the oxidation of water. Two key homologous 32 KD protein subunits, D1 and D2, in PSII are transmembrane proteins and are at the heart of the PSII complex. It has been said of PSII that ""Of all the biochemical inventions in the history of life, the machinery to oxidize water — photosystem II — using sunlight is surely one of the grandest." (Sessions, A. et al, Current Biology 19 (2009)

    The net reaction carried out by PSII is the oxidation of water and reduction of plastoquinone.

    2PQ + 2H2O → 2PQH2 + O2 (g)

    The oxidation number of oxygen in water is -2 and 0 in O2, so this is clearly a loss of electrons or oxidation of the water. Note that water is not converted to 2H2 + O2, as in the electrolysis of water. Rather the Hs are removed from water as protons in the lumen of the cholorplast, since the part of PSII which oxides water is near the lumenal end of the transmembrane complex. Protons required to protonate the reduced (anionic) form of plastaquinone to form PQH2, an activity of PSII found closer to the stroma, derive from the stroma. That being said, researchers actively trying to develop a photosynthetic scheme or mimic that does produce H2 for use as a clean and essentially boundless fuel source to replace climate-warming fossil fuels.

    A quick look at standard reduction potentials (SRP) shows that the passing of electrons from water (dioxygen SRP = +0.816 V) to plastoquinone (approx SRP of 0.11 ) is not thermodynamically favored. The process is driven thermodynamically by the energy of the absorbed photons.

    The crystal structure of PSII from a photosynthetic cyanobacterium consists of 17 polypeptide subunits with metal and pigment cofactors and over 45,000 atoms. Of particular interest is the P680 chlorophyll reaction center, which consists of four monomeric chlorophylls adjacent to a key Tyr 161 side chain. When H2O gets oxidized to form dioxygen, 4 electrons must be remove by photoactivated P680. In PSII, this process occurs in 4, single electron steps, with the electrons first being transferred to the oxygen evolving complex. The electrons passed through the Mn complex are delivered to P680 by a photoactive Tyr 161 (Tyr Z or YZ) free radical.

    Five discrete intermediates of the OEC, S0-S4, are suggested from the experimental data and are consistent with the Kok cycle, which we will discuss below. These were postulated from experiments in which spinach chloroplast were illuminated with short light pulses. A pattern of dioxygen release was noted that repeated after 4 flashes. Ultimately, light absorption by P680 forms excited state P680*, which donates an electron to pheophytin, which passes them to quinones. Hence P680 gets photoxidized as it forms the cationic P680+. This then removes an electron from Tyr 161 (YZ) producing the tryosine radical cation, Tyr 161.+. Given its positive charge, it reactive nature as a free radical, and its proximity to Mn ions in the OEC, it pull an electron from a Mn ion in the OEC. Ultimately the net effect after this processes repeats itself 4 times is the oxidation of two H2Os, which injects 4 electrons back into the OEC to return to the basal state.

    The mechanism is very complicated and still not fully understood. It is perhaps easiest to think about the mechanism involving a series of sequential electron and proton transfer and their accompanying change in charge and redox states. Most biochemistry students have a limited understanding of transition state complexes and chemistry but even the experts struggle with the mechanism.

    In summary, for PSII in plants

    1. a pair of chlorophylls (P680) in the D subunits absorb light (maximum absorbance around 680 nm) and reach an excited state
    2. electron transfer from P680 to a nearby chlorophyll with a lower energy level for the excited state electron occurs, which produces an anionic chlorophyll. This chlorophyll has 2 H+ ions in the chlorophyll instead of Mg2+ (again note the charge balance). After electron transfer, P680 now becomes the cation P680+.
    3. This "anionic" chlorophyll transfers an electron to oxidized plastoquinone.
    4. The P680+, a strong oxidizing agent, removes one electron from an adjacent Tyr 161 to reform P680 and the radical cation Tyr 161.+. Its proximity to the OEC complex leads to it removing an electron from the OEC, making it a more potent oxidizing agent
    5. This process repeats a total of 4 times to fully oxidize two water molecule to produce 1 O2 with the 4 electrons removed from 2 waters added back to the metal centers of the OEC.

    This suggests that there are 5 states of the OEC, an initial state, which we will call S0, and four other states (S1, S2, S3 and S4). S1 forms after removal of one electron from the OEC by the adjacent radical cation Tyr 161.+ (formed after absorption of one photon). S2, S3 and S4 are sequentially formed after removal of one electron by a newly regenerated Tyr 161.+ after another round of photoexcitation. S4 then returns to its original state, S0. These series of reaction are called the Kok cycle, which is shown in Figure \(\PageIndex{3}\) below.

    KokCyclePPTXSlide-01.svg
    Figure \(\PageIndex{3}\): The Kok cycle

    There is no structural information given in the above figure. What is shown instead are possible and consistent oxidation numbers of the four Mnn+ ions in the OEC that are consistent with charge balance and the changes in the oxidation number (-2) of the oxygen atom in water as it progress to O2 with an oxidation number of 0. The Mn ion states in the Kok diagram denote different discrete oxidation states where n is the number of oxidative “equivalents” stored in the OEC during cycle progression. Think of the OEC as the key catalyst, which will interact with substrate H2O molecules. We start with the S0 state and must return to it in the full cycle.

    Remember that when O2 acts as an oxidizing agent in combustion reactions, it forms 2H2O. That requires the addition of four electrons. If done sequentially, the oxygen intermediates include superoxide, peroxide, and oxides, which when protonated is water. Hence two waters and four cycles are required to remove the four electrons required to produce dioxygen. Intermediate but transient oxygen states are also presumably important in this mechanism.

    A similar mechanism is found in PSI, except plastacyanin, not dioxygen is oxidized, with electrons moved to ferrodoxin. This is likewise a difficult process since the reduction potential for oxidized plastocyanin (the form that can act as a reducing agent) is +0.37 while for ferrodoxin it is -0.75. This transfer of electrons is an uphill thermodynamic battle since the more positive the standard reduction potential, the better the oxidizing agent and the more likely the agent becomes reduced. What drives this uphill flow of electrons. Of course, it is the energy input from photon. We won't go into any more detail about PSI since it is very similar to PSII but of course does not have the OEC.

    The Oxygen Evolving Complex - OEC

    Even though this is not a bioinorganic textbook, we must move past the "simple" Kok cycle diagram and look at the actual structure of the minicatalyst, the OEC) and the protein and water (substrate) environment around it to understand the mechanism. The mechanism of the OEC is still not fully understood. It's experimentally difficult to unravel given its complexity as the intermediates are very labile and the x-ray induced transient alterations in the structure of OEC complicate matters more. Paradoxically it is quite simple in overall terms. Here is the essential reaction:

    2H2O + 4 photons → 4 H+(lumen) + 4 e- + O2.

    The crystal structure of PS2 from T. vulcanus has significantly improved our understanding of the OEC and electron flow on water oxidation. We will concentrate on developing an understanding of the amazing Photosystem II from Thermosynechococcus vulcanus, a cyanobacteria (19 subunits with 35 chlorophylls, two phenophtyins, 11 beta cartotenes, 2 plastoquinones, 2 heme irons, 1 non heme iron, 4 Mn ions, 3-4 Ca ions, 3 Cl ions, 1 carbonate ion, and around 2800 water molecules).

    Nature has appeared to evolve a single gene for the central protein in PSII that binds the OEC. The cluster, Mn4CaO5, appears identical in all photosynthetic organisms, and is shown below. Researchers were surprised to find that the Ca ion was an integral part of the basic geometric “framework” of the OEC instead of a Mn which was found to be “dangling” from the basic geometric framework. A detail structure of the OEC from T. vulcanus is shown in Figure \(\PageIndex{4}\) below.

    3WU2_OEX5ASess8-OEX4H2Os_Alone.png
    Figure \(\PageIndex{4}\): Structure of the OEC

    Note the basic structure is distorted cube the metal ions at every other corner separated by oxides. Again, it was surprise that not all of the 4 Mn2+ ions were in the cubic structure. Note one "dangling" Mn2+ with the other last metal site in the distorted cube occupied by Ca2+. Four oxygens (presumably from waters) are shown interacting with MN4 and CA1.

    It is very difficult to visualize this structure correctly from a 2D figure. Figure \(\PageIndex{5}\) below shows an interactive iCn3D model of the OEC with bound water of photosystem II from Thermostichus vulcanus (3WU2) which should help in visualizing this structure. (very long load time!)

    OEXandHOH_photosystem II_Thermostichus vulcanus_3WU2.png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{5}\): OEX with bound water of photosystem II from Thermostichus vulcanus (3WU2). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...F8vwgkDXV6C1S6. (very long load time)

    The shape outlined by O5-CA-O1-MN1 and MN3-O2-MN2-O3 is similar to cubane.

    Now let's zoom out and view some of the amino acid side chains that interact with the OEC from T. vulcanus. These are shown in Figure \(\PageIndex{6}\) below.

    3WU2_OEX5ASess6.png

    Figure \(\PageIndex{6}\): OEC and surrounding amino acids from T. vulcanus

    The coordination number for all the Mn ions (including those interacting with waters) is identical. Note the proximity of Tyr 161 which is involved in electron removal from the OEC after its becomes the radical cation Tyr 161.+ in the primary photoxidation event.

    Figure \(\PageIndex{7}\) below shows an interactive iCn3D model of the OEX with surrounding amino acids and water in photosystem II from Thermostichus vulcanus (3WU2). (long load time) presented to once again help you better understand the 2D structure shown above.

    OEX with surrounding amino acids and water in photosystem II from Thermostichus vulcanus (3WU2).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{7}\): OEX with surrounding amino acids and water in photosystem II from Thermostichus vulcanus (3WU2). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...bP6ttbaWUmQAg7

    The OEC can be thought of as a distorted cubane with a MN3-O4-MN4-O5 back. Bonds to O5 are longer than the other bonds which suggest they they are weaker than the other metal-oxygen bonds. This could suggest that it may not be an oxo (O2-) ligand but another variant such as OH- (lower charge), which may imply involvement in the splitting of dioxygen in the reaction mechanism. From a mechanistic perspective, an O-O bond must form between two waters. Both sets of bound "waters" (purple spheres in Figure 4 and red spheres in Figure 5 shown without Hs attached if they are present) are close to O5.

    It is important to remember than electrons removed from the metal ions in the OEC by Tyr 161.+ must be restored to the OEC to allow the catalytic cycle to continue. These electrons come from the waters that get oxidized. Such a reversible loss and gain of electrons most readily occurs from the transition state Mn ions, which you all remember from introductory chemistry have multiple oxidation states. To bring back introductory chemistry again, we present the standard reduction potentials oof different Mn ions in Table \(\PageIndex{1}\) below.

    Reduction reaction Standard Reduction Potential
    Mn2+ (aq) + 2 e-→ Mn (s) -1.185
    MnO4- (aq) + 2 H2O (l) + 3 e- → MnO2 (s) + 4 OH- +0.595
    MnO2(s) + 4H+ + e- → Mn3+ + 2H2O +0.95
    MnO2(s) + 4H+ + 2e- → Mn2+ + 2H2O +1.23
    MnO4- (aq) + 8 H+ (aq) + 5 e- → Mn2+ (aq) + 4 H2O (l) 1.507
    MnO4- (aq) + 4 H+ (aq) + 3 e- → MnO2 (s) + 2 H2O (l) 1.679
    HMnO4- + 3H+ + 2e- → MnO2(s) + 2H2O +2.09
    O2(g) + 4H+ + 4e- → 2H2O +1.229 +1.229

    Table \(\PageIndex{1}\): Standard reduction potentials for Mn ions compared to O2.

    You should be able to determine the oxidation number of the Mn ion in each compound. Based on standard reduction potentials, which oxidation states might be sufficient for the oxidation of H2O in the OEC?

    How does this translate into structural/chemical changes in the OEC? Figure \(\PageIndex{8}\) below provides a recent mechanism consistent with each of the Kok states (S0-S4).

    OEC_Mechanism040522.svg
    Figure \(\PageIndex{8}\): Mechanism for water oxidation and O2 formation through the OEC consistent with the Kok cycle (adapter from Britt & Marchiori, 10.1126/science.aaz4522)

    The proposed change in redox state for each Mn ion is illustrated with Mn (III) ions in purple and Mn (IV) ions in yellow. Note the change in oxidation state of the 4 Mn ions from (III, IV, III, and III) in S0 to (IV, IV, IV, and IV) for all of them in S3 and S4. A flip in the side chain of E180 in S2 allows the binding of Mn1 through an oxy link to Ca. ​​ There are many possible differ forms of oxygens in the structure including waters, oxides (bridging oxos and possibly terminal oxides) and hydroxides and the exact form at some sites is still a bit uncertain. Note also the elegance of having a Mn4 cluster to catalyze the 4 electron oxidation of 2 water through the loss of 4 electrons. Also the redox state change in the Mn ions is different than the one shown in the Kok diagram in Figure 3. In addition the final O2 producing step going from S4 → S0 is still uncertain. The above mechanism is based on x-ray structures of intermediates and quantum calculations. In it S4 has an Mn(IV)O. that bonds with the bridging O5 to form O2.

    Waters

    As water is a reactant in PSII, there must water channels leading to the OEC that provide a way for water to enter and for protons to be removed and directed to the lumen to develop a proton gradient. Another rendering of the Kok cycle, the position of the OEC in PSII in the luminal side of the membrane, and the presence of water channels (Cl1, O4, O1) and the Yz network, which connect Tyr 161 (Yz) to the lumen, are shown in Figure \(\PageIndex{9}\) below.

    waterandprotonchannelsPSII_Fig1A.svg
    Figure \(\PageIndex{9}\): An overview of Photosystem II and the main water channels and networks from the OEC to the lumenal side. The left part of the figure illustrates the structure of PS II showing the membrane-embedded helices and the extrinsic subunits in beige. The OEC and the water channels, in addition to the Yz network, are shown in color. The Kok cycle of the water oxidation reaction that is triggered by the absorption of photons is shown on the right and highlighted with a blue circle. F represents a photon. Hussein, R., Ibrahim, M., Bhowmick, A. et al. Structural dynamics in the water and proton channels of photosystem II during the S2 to S3 transition. Nat Commun 12, 6531 (2021). https://doi.org/10.1038/s41467-021-26781-z. Creative Commons Attribution 4.0 International License. http://creativecommons.org/ licenses/by/4.0/.

    A more detailed representation of water channels and the Yz networks are shown in Figure \(\PageIndex{10}\) below.

    waterandprotonchannelsPSII_Fig1b.svg
    Figure \(\PageIndex{10}\): A detailed view of the water channels showing the waters within each channel (O1 Channel red dotted, O4 Channel blue dotted, and Cl1 Channel green dotted). The region highlighted with solid yellow represents the Yz network. Residues involved in forming bottlenecks in the channels are shown in black. Water molecules found in the crystal structures are labeled with blue numbers with the channel. Hussein, R., Ibrahim, M., Bhowmick, A. et al., ibid.

    These structures show that there is no direct water pathway from across the OEC and that all channels restrict water movement to some degree. The O4 and Cl1 channels are narrower than the O1 channel so water in those is less mobile. In the O4 channels, waters 50-53 are near charged groups and are close to a major bottleneck (residues D1-N338, D2-N350, and CP43-P334, -L334).

    Based on the x-ray structures and molecular dynamic simulations, it appears that the O1 channels allows access of water to the OEC. The Cl1 channel A, which is more rigid, may be involved in H+ transfer during S2 → S3.

    The Last Steps: Electron Transfer to Plastoquinone

    We are almost ready for the next section in which we will present the flow of electrons away from PSII through mobile electron carriers, leading to the synthesis of NADPH for reductive biosynthesis of carbohydrates. Before we leave PSII, let's look at what happens to the radical anion P680-, also know as PheA- (pheophytin A) or PheAD1, the reaction center chlorophyll without a central Mg2+ ion) which received an electron from photooxidation of the reaction center P680, as summarized again in Figure \(\PageIndex{11}\) below.

    etransfer_RxCenter.svg
    Figure \(\PageIndex{11}\): The reaction center electron transfer reactions in PSII.

    Pheophytin A- passes its electron to plastoquione A (in PSII). which passes it on to the lipophilic mobile electron carrier in the thylakoid membrane plastoquinone. It is similar to the mobile electron carrier in mitochondrial electron transport, ubiquinone. Ultimately these are passed to NADP+ to form NADPH for reductive biosynthesis.

    Figure \(\PageIndex{12}\) below shows an interactive iCn3D model highlighting just the OEX, pheophytin A (PHO) and plastoquinone A (PL9) in photosystem II from Thermostichus vulcanus (3WU2). (long load time).

    OEX,_(PHO) _plastoquinone A (PL9)PS II Thermostichus vulcanus (3WU2).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{12}\): the OEX, pheophytin A (PHO) and plastoquinone A (PL9) in photosystem II from Thermostichus vulcanus (3WU2). (long load time). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...H7Mycbx1PsFbt6

    The OEX (OEC) is shown in spacefill, CPK colors, PHO is shown in spacefill magenta and PL9 in spacefill cyan. Note the proximity of PHO and PL9 for easy electron transfer to plastoquionone A.

    Given the number and proximity of high energy reactive species in the reaction center, it should not be surprising that side reaction can occur. These would decrease the efficiency of light energy transduction and also could damage molecular components of PSII (and similarly PSI)

    Figure \(\PageIndex{13}\) belows shows a standard reduction potential diagram for the P680 - Pheophytin A - PlastoQuinone A triad (abbreviated P Phe Q) in PSII.

    Enhancing photosynthesis in plants the light reactionsFig3.svg
    Figure \(\PageIndex{13}\): Charge recombination pathways in PSII (The energy gaps are not drawn to scale). Cardona T, Shao S, Nixon PJ. Enhancing photosynthesis in plants: the light reactions. Essays Biochem. 2018;62(1):85-94. Published 2018 Apr 13. doi:10.1042/EBC20170015Creative Commons Attribution License 4.0 (CC BY).

    Safe routes for charge recombination between P+ and QA are indicated in blue, the damaging route producing 1O2 in red and radiative pathway in green. Stabilization of the P+PheoQA state helps prevent reverse electron flow to form P+PheoQA and subsequent charge recombination to form PPheoQA. For clarity, the details of the additional electron transfer steps, including the oxidation of water and the reduction of plastoquinone to plastoquinol by PSII, collectively termed as photosynthesis, are omitted. Abbreviations: P, primary electron donor of PSII; Pheo, pheophytin electron acceptor; QA, primary plastoquinone electron acceptor; 1O2, singlet oxygen; 3O2, triplet oxygen; 3P, triplet excited state of P.


    20.2: The Kok Cycle and Oxygen Evolving Complex of Photosystem II is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.