Molecular Modelling Studies of the Oxygen Evolving Complex in Photosynthesis

1. Introduction

Photosynthesis is a biological process at which occurs in the thylakoid membrane. This process occurs in some organisms, such as cyanobacteria, algae and plants. It is induced by light or solar energy. It also produces chemical energy which is converted from light in chloroplast. An electron from ground state energy level will be excited to an excited energy level by obtained energy from light. For most cases, after electron excites, it will rebounds to the ground state energy level  again and the absorbed energy will be converted. In other case which involve electron acceptor nearby, excited electron moves from electron donor compound into electron acceptor compound. This transition causes positive charge on ‘donor’ compound, while causes negative charge on the ‘acceptor’ compound. This transition is known as photoinduced charge separation and takes place in the reaction centre. High-energy of electrons are transported through two Photosynthetic Reaction Centre called Photosystems, Photosystem I and Photosystem II. Photosynthesis process is started by Photosystem II captures light and electron excites to the excited energy level. It will oxidize oxygen from H₂O (-2 charge) into O₂ (0 charge). In Photosystem II, the core complexes are water molecules as a donor side and quinones as a acceptor side. In Photosystem II there are two type of quinones, Q_A and Q_B. They catalyze electrons transfer from water to plastoquinone, transport protons to the lumen, produce oxygen gas and release produced plastoquinol into the membrane phase. Excited electron from Photosystem II will move into Photosystem I, which is more complex than Photosystem II, and generates reducing power. Due to proton gradient in this process, ATP will be synthesized by ATP synthase from ADP and it becomes cellular energy.^[1]

Previous research’s focus is on the Photosystem II Reaction Centre which undergoes reduction of quinone to forms semiquinone and finally quinol. Recently, X-ray data can not differentiate plastoquinones and plastosemiquinone (one-electron reduced) forms. It is assumed that the amount and strength of hydrogen bonds in the plastosemiquinone form become the main factor in electron transfer characteristic. Therefore, there are some important informations which is required in determining its electron transfer characteristic. Firstly, Electron Paramagnetic Resonance (EPR) is used to determine spin density distribution and hydrogen bonding of semiquinone. Secondly, Density Functional Theory is used to interpret of hyperfine couplings and to predict accurate spin density distribution. Finally, ONIOM study is recently used to combine two or more computational techniques which will make it appropriate to analyse some phenomenon in a large system with high precision. In this article, authors use ONIOM (Quantum mechanics/Molecular Mechanics) to calculate the spin density distribution for the plastosemiquinone anion radical in the Q_A binding site of Photosystem II. A large number of models were examined to explore the effect of iron depletion on the Q_A site semiquinone spin density distribution and resultant hyperfine couplings. The calculated spin density in the Q_A site model which has divalent metal ion in the non-heme site indicates that differential hydrogen bonding strength to the O1 and O4 of the radical results in an asymmetric spin density distribution in the semiquinone anion free radical form, which the hydrogen bond to the proximal O1 is stronger.^[36]

2. Basic Principle
   1.            Photosynthesis

Photosynthesis is a very useful process for plants which can produces food for itself. To obtain energy or food, plants convert solar energy into chemical energy. This process has a simple reaction scheme which associates a light-induced reaction between carbon dioxide (CO₂) and water to forms carbohydrate and oxygen molecule.^[1] The overall reaction can be drawn as a equation below :

However, photosynthesis mechanism is complex and contains large number of proteins and small molecules. The mechanism starts when lights or photons are captured by chlorophylls as a pigment in chloroplast. This photon energy will excites electron and generates high-energy electron which has high reducing potential. This potential will reduce NADP⁺ into NADPH and produce ATP. This process is called as a ‘light reaction’. Then, obtained NADPH and ATP from ‘light reaction’ reduce carbon dioxide and form 3—phosphoglycerate. This process is called as a ‘dark reaction’.^[1]

Figure 1 The ‘Light Reaction’ of Photosynthesis^[1]

2.2              Photosystem II

In green plants, two complexes mediates photosynthesis process, Photosystem I and Photosystem II.^[1] Photosystem I and Photosystem II are complexes where ‘light reaction’ is catalyzed. Photosystem complexes are exist in thylakoid membrane. In the first step, electrons from water (donor side) are utilized by Photosystem II to reduce plastoquinone to forms plastosemiquinone. This plastosemiquinone is reduced for the second time to forms plastoquinol and then this plastoquinol is distributed into the membrane and recovered by another fresh plastoquinone from plastoquinone pool^[2].

Figure 2 Process in Chloroplast which produces chemical energy from light energy^[1]

2.3              Transfer of electron in Photosystem II

The first step in photosynthesis is photoreceptor molecule absorps light energy. In the green plants, the photoreceptor molecule is called chlorophyll a, a substituted tetrapyrrole which has four nitrogen atoms integrated to a magnesium ion.

In order to distribute photons to the reaction centre, the number of pigments performing excitation energy transfer is large so photons can be ‘harvested’ effectively.^[8,9] Within Photosystem II core complex, transfer of the excitation energy from antenna to the reaction centre is approximately 40-50 ps.^[10,3,11,12] It is slower compared to the different path, from outer antenna into core complex. This condition is caused by chlorophyll-reaction centre’s distance which quite large.

Electron transfer occurs in a reduction of quinone into the quinol which involve Q_A and Q_B. Electron transfer travels to the membrane plane perpendicularly. At the Q_A site, it changes from perpendicular into parallel toward Q_B site.

Figure 3 Photoinduced Charge Separation in Photosynthesis^[1]

3. Literature Review
   1.            Quinones

Quinones are cofactors which very important in photosynthesis. They involve in the initial charge separation as an electron acceptors. There are two type of quinone in Photosystem II reaction centre, Q_A and Q_B. However, Q_C was discovered in the crystallized dimeric Photosystem II core complex recently. Pheophytin anion radical reduces Q_A, one-electron quinone, to forms semiquinone anion radical. After that, electron transfer to Q_B will oxidizes semiquinone anion radical while forms Q_B semiquinone anion radical. This process is repeated for the second time. Thus, this Q_B now can obtains two protons to neutralize its negative charge (-2). This form called quinol (QH₂) and will leaves Q_B site. This quinol will be removed and recovered by new quinone.

Figure 4 Electron chains in reduction process of Q_B^[1]

The electron from reduced quinone are transported to the soluble cytochrome c intermediate, called cytochrome c₂. However, instead of obtaining electron from this cytochrome, Photosystem II obtains electron from water and generates oxygen molecule. This reaction occurs at special centre contains four manganese ions.^[1] Overall reaction which involve reduction quinone can be written in equation below :

3.2              Complexes of Iron-Quinone Acceptor

Photosystem II reaction centre has non-heme iron at its acceptor side. Different from heme iron, this non-heme iron has distorted octahedral geometry even though it is also hexacoordinate. Earlier discovery on spinach’s chloroplast showed that at pH 7.8 component at both Q_A and Q_B has midpoint potential about 360 mV.^[13] This component is pH-dependent at which pH 6.0 to 8.0, its redox potential’s range will be approximately from 450 to 360 mV.^[14] Electron Paramagnetic Resonance can identifies this component, and now it is known as an iron-quinone acceptor complex.^[15]

According to an experiment, there is a proof that Photosystem II activity requires bicarbonate. There is a significant restoration decrease of electron transfer rate obtained when samples are flushed by nitrogen gas or CO₂-depleted air with high concentrated of formate and acetate. In Photosystem II core complex, there is only one bicarbonate molecule that had been successfully identified so far, which is the attached to the non-heme iron. It gives ‘bicarbonate effect’ to the acceptor side.^[16] There are two indications that bicarbonate displaces NO as a non-heme iron’s ligand. Firstly, slowing electron transfer between Q_A and Q_B is reversed. Secondly, if  NaHCO₃ is added, the characteristic of EPR signal from the Fe²⁺-NO adduct will be reduced.^[17] Using Fourier Transfrom Infrared (FT-IR) Fe²⁺/Fe³⁺ difference spectroscopy, Hienerwadel and Berthomieu concluded that bicarbonate is a ligand which is integrated to the non-heme iron similar to the bidentate ligand, it changes into monodentate ligand and does not deprotonated upon iron oxidation. While iron oxidation deprotonates histidyl ligand integrated to the non-heme iron.^[18] However, according to different experiment using electrostatic computations, Ishikita and Knapp concluded that pKa shifts of groups outside the iron’s first coordination shell can be alleged from the pH dependence of the redox potential.^[19]

Figure 5 Non-heme iron in Photosystem II Reaction Centre^[1]

3.3              Electron Transfer in Quinone

Quinol formation from quinone needs two steps, both two electrons and protons. Firstly, a semiquinone anion radical (Q_B•–) is stabilized by a protonation of nearby amino acid side chains. Some previous research proposed that coupling between Q_B• and Q_A is sufficiently strong and able to generates proton uptake near Q_B by the reduction of Q_A^{[20,21,22,23]}. It is also able to adjusts the ΔG₀ for electron transfer. After electron reach Q_B, further both proton uptake and rearrangements arise. In Photosystem II core complex, there are several conclusions associated to electron transfer from Q_A•– to Q_B. Firstly, it is heterogeneous and must be defined with both 0.2 – 0.8 ms and 2 – 3 ms constant.^[25] Secondly, it is pH-dependant^[25,26,27], H₂O/D₂O exchange^[26,28,29] and dehydration^[30,31] indicating a coupling to proton transfer and/or water dependent structural relaxation process. Thirdly, it is coupled to protein dynamics and shows a marked temperature dependence being completely prevented below 200 K.^{[31,32,33,34]} . The last discovery is that according to an electron tunneling analysis, the overall reaction between the quinones is much slower than expected. It indicates electron transfer is not rate limiting.^[33,35] While electron transfer to the Q_B (second step) is coupled to the first proton distribution.

4. Previous Research

In the previous research, authors used a hybrid method called Our own N-layered Integrated Molecular Orbital and Molecular Mechanics (ONIOM) method. It combines two or more computational techniques in one calculation. This method is appropriate to examine the chemistry aspects from large number of systems with high precision. It is also identifies the effects of the specific quinone binding site up to radius of 20 Å. ^[36]

Table 1 Optimized Plastosemiquinone and Plastoquinone (in bracket) Geometries (in Å)^[36]

For model 1 and 4, there is a divalent ion, while in model 2 and 3 divalent ion is not exist. From data above, it can be seen that for model 1 and 3, lengthening of the carbonyl bond length from 0.04 Å to 0.05 Å is formed by the reduction of quinone to  the semiquinone. It also can be seen that the presence or absence of a divalent ion in the non-heme site affects plastosemiquinone O1–Hydrogen bond distance. For model 1 and 4, their hydrogen bond is shorter compared to the hydrogen bond in model 2 and 3. This condition suggests that the existence of divalent ion attached to the non-heme site increases hydrogen bond’s strength. ^[36]

Figure 6 Mulliken spin population for plastosemiquinone in (a) isolated, (b) model 1, (c) model 2, (d) model 3, (e) model 4, and (f) model 5^[36].

From Figure 6, it can be seen that models 1 to 5 representation show the Mulliken spin population shifts from the isolated radical to the protein binding site. From the previous discovery, it had been found that spin population from the oxygen atom’s position redistributes to the carbon of the carbonyl group as the primary effect of hydrogen bonding to the semiquinone’s oxygen atom. In the model 1 (b), it can be seen that Mulliken spin population at C1 and O1 are 0.17 and 0.18 respectively, while at C4 and O4 are 0.10 and 0.25 respectively. Small difference in spin population indicates that redistribution occurs between C1 and O1 but not occurs between C4 and O4. For model 2, the redistribution occurs at O4 and C4 but not at C1 and O1. For model 3, the redistributions are inspected for both carbonyl groups. For model 4, the redistribution is occurs only at C1 and O1, similar to the model 1. For both model 1 and 4, the presence of divalent ion at the non-heme site makes hydrogen bond formation to the O1 atom becomes stronger. It is proven by the shorter hydrogen bond, which can be seen in Table 1. This stronger hydrogen bond causes hydrogen bond to the O4 atom becomes weaker and produces a significant polarization of the C1-O1 spin population, with a spin population on O1 decline from 0.25 to 018 while spin population on C1 increase from 0.07 to 017. The increased spin population at C1 causes lower spin population at C2 and C6 atoms, while in turn causes higher spin population at C3 and C5 atoms. By removing the divalent ion (model 2), the spin population redistribution is now reversed. Spin population at O4 is decreased from 0.25 to 0.21, while at C4 increases from 0.09 to 0.16. Now, hydrogen bond at the O4 atom is stronger than at O1 and the polarization of the spin population is reversed. ^[36]

Quantum/molecular mechanics studies of the Q_A site in the Photosystem II acknowledge that obtained spin density distribution of the plastosemiquinone is very sensitive to the occupation of the non-heme iron site. If divalent ion is attached to the non-heme iron site, it causes a stronger hydrogen bond interaction and causes polarization of the spin population on the Q_A plastosemiquinone. While the absence of divalent ion causes an opposing spin population’s polarization and reduction of the spin population. Removal of the bicarbonate ligand followed by divalent ion gives more symmetrical spin population distribution at both carbonyl.

From previous research which was done in 1997, using EPR-II basis set and B3LYP functional, bond distances had been calculated between semiquinone and 4 solvents ; water, methanol, ethanol, and isopropanol. All calculation was done in two conformation of the solvents, an eclipsed conformation (A) and staggered conformation (B). Both conformation has the same model in their structure, at which the quinone is surrounded by 4 molecules of solvents only.

Table 2 Calculated Bond Distances for the Complexes Between Quinone and Solvents (in Å)^[37]

From the table above there is no significant changes in bond distances between carbon-oxygen or carbon-carbon bond from both conformations. However, there is slightly change (0.01 – 0.06 Å) in hydrogen bond distance from both conformations. Overall, conformation B has longer hydrogen bond than conformation A. It was assumed that conformation A is more stable than conformation B as staggered conformation has steric factor than eclipsed conformation.

(a)                                                           (b)

Figure 7 Example of the Conformation A (a) and Conformation B (b) Modelling Between Quinone and Water Molecule

5. Objective

In this research project, the main objective is to determine the effect of the hydrogen bonding on the spin densities and hyperfine coupling of the para-Benzosemiquinone anion radical in water and alcohol solutions using hybrid density functional studies.

In order to achieve this objective, there are some steps that must be done. First of all, the optimized geometry between semiquinone and solvents must be constructed. Therefore, bond distances and the coordinates of the atoms will be obtained. From the optimized geometry coordinates, spin densities and hyperfine coupling can be obtained. To achieve all of this data, EPR-II basis set and both B3LYP and bp functional will be used. Two functional will be used to identifies whether there are some differences or not. Similar to the previous research, two conformations will be constructed. Compared to the previous research which constructed molecular modeling between semiquinone and four solvent molecules, this research will be developed by using lots of solvent molecules due to the fact that in reality there are lots of solvent molecules, not only four molecules of solvent.

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