Chlorophyll Pair Research Articles

Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I.

Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.

The synergy between the PscC subunits for electron transfer to the P840 special pair in Chlorobaculum tepidum

The primary photochemical reaction of photosynthesis in green sulfur bacteria occurs in the homodimer PscA core proteins by a special chlorophyll pair. The light induced excited state of the special pair producing P840+ is rapidly reduced by electron transfer from one of the two PscC subunits. Molecular dynamics (MD) simulations are combined with bioinformatic tools herein to provide structural and dynamic insight into the complex between the two PscA core proteins and the two PscC subunits. The microscopic dynamic model involves extensive sampling at atomic resolution and at a cumulative time-scale of 22µs and reveals well defined protein–protein interactions. The membrane complex is composed of the two PscA and the two PscC subunits and macroscopic connections are revealed within a putative electron transfer pathway from the PscC subunit to the special pair P840 located within the PscA subunits. Our results provide a structural basis for understanding the electron transport to the homodimer RC of the green sulfur bacteria. The MD based approach can provide the basis to further probe the PscA-PscC complex dynamics and observe electron transfer therein at the quantum level. Furthermore, the transmembrane helices of the different PscC subunits exert distinct dynamics in the complex.

Charge transfer as a mechanism for chlorophyll fluorescence concentration quenching

Highly concentrated solutions of chlorophyll display rapid fluorescence quenching. The same devastating energy loss is not seen in photosynthetic light-harvesting antenna complexes, despite the need for chromophores to be in close proximity to facilitate energy transfer. A promising, though unconfirmed mechanism for the observed quenching is energy transfer from an excited chlorophyll monomer to a closely associated chlorophyll pair that subsequently undergoes rapid nonradiative decay to the ground state via a short-lived intermediate charge-transfer state. In this work, we make use of newly emerging fast methods in quantum chemistry to assess the feasibility of this proposed mechanism. We calculate rate constants for the initial charge separation, based on Marcus free-energy surfaces extracted from molecular dynamics simulations of solvated chlorophyll pairs, demonstrating that this pathway will compete with fluorescence (i.e., drive quenching) at experimentally measured quenching concentrations. We show that the rate of charge separation is highly sensitive to interchlorophyll distance and the relative orientations of chromophores within a quenching pair. We discuss possible solvent effects on the rate of charge separation (and consequently the degree of quenching), using the light-harvesting complex II (LH2) protein from rps. acidophila as a specific example of how this process might be controlled in a protein environment. Crucially, we reveal that the LH2 antenna protein prevents quenching, even at the high chlorophyll concentrations required for efficient energy transfer, by restricting the range of orientations that neighboring chlorophyll pairs can adopt.

LHCA4 residues surrounding red chlorophylls allow for fine-tuning of the spectral region for photosynthesis in Arabidopsis thaliana.

Improving far-red light utilization could be an approach to increasing crop production under suboptimal conditions. In land plants, only a small part of far-red light can be used for photosynthesis, which is captured by the antenna proteins LHCAs of photosystem I (PSI) through the chlorophyll (Chl) pair a603 and a609. However, it is unknown how the energy level of Chls a603-a609 is fine-tuned by the local protein environment in vivo. In this study, we investigated how changing the amino acid ligand for Chl a603 in LHCA4, the most red-shifted LHCA in Arabidopsis thaliana, or one amino acid near Chl a609, affected the energy level of the resulting PSI-LHCI complexes in situ and in vitro. Substitutions of the Chl a603 ligand N99 caused a blue shift in fluorescence emission, whereas the E146Q substitution near Chl a609 expanded the emission range to the red. Purified PSI-LHCI complexes with N99 substitutions exhibited the same fluorescence emission maxima as their respective transgenic lines, while the extent of red shift in purified PSI-LHCI with the E146Q substitution was weaker than in the corresponding transgenic lines. We propose that substituting amino acids surrounding red Chls can tune their energy level higher or lower in vivo, while shifting the absorption spectrum more to the red could prove more difficult than shifting to the blue end of the spectrum. Here, we report the first in vivo exploration of changing the local protein environment on the energy level of the red Chls, providing new clues for engineering red/blue-shifted crops.

Triplet states in the reaction center of Photosystem II.

In oxygenic photosynthesis sunlight is harvested and funneled as excitation energy into the reaction center (RC) of Photosystem II (PSII), the site of primary charge separation that initiates the photosynthetic electron transfer chain. The chlorophyll ChlD1 pigment of the RC is the primary electron donor, forming a charge-separated radical pair with the vicinal pheophytin PheoD1 (ChlD1+PheoD1-). To avert charge recombination, the electron is further transferred to plastoquinone QA, whereas the hole relaxes to a central pair of chlorophylls (PD1PD2), subsequently driving water oxidation. Spin-triplet states can form within the RC when forward electron transfer is inhibited or back reactions are favored. This can lead to formation of singlet dioxygen, with potential deleterious effects. Here we investigate the nature and properties of triplet states within the PSII RC using a multiscale quantum-mechanics/molecular-mechanics (QM/MM) approach. The low-energy spectrum of excited singlet and triplet states, of both local and charge-transfer nature, is compared using range-separated time-dependent density functional theory (TD-DFT). We further compute electron paramagnetic resonance properties (zero-field splitting parameters and hyperfine coupling constants) of relaxed triplet states and compare them with available experimental data. Moreover, the electrostatic modulation of excited state energetics and redox properties of RC pigments by the semiquinone QA- is described. The results provide a detailed electronic-level understanding of triplet states within the PSII RC and form a refined basis for discussing primary and secondary electron transfer, charge recombination pathways, and possible photoprotection mechanisms in PSII.

Requirement of Chloride for the Downhill Electron Transfer Pathway from the Water-Splitting Center in Natural Photosynthesis.

In photosystem II (PSII), Cl- is a prerequisite for the second flash-induced oxidation of the Mn4CaO5 cluster (the S2 to S3 transition). We report proton transfer from the substrate water molecule via D1-Asp61 and electron transfer via redox-active D1-Tyr161 (TyrZ) to the chlorophyll pair in Cl--depleted PSII using a quantum mechanical/molecular mechanical approach. The low-barrier H-bond formation between the substrate water molecule and D1-Asp61 remained unaffected upon the depletion of Cl-. However, the binding site, D2-Lys317, formed a salt bridge with D1-Asp61, leading to the inhibition of the subsequent proton transfer. Remarkably, the redox potential (Em) of S2/S3 increased significantly, making electron transfer from S2 to TyrZ energetically uphill, as observed in Ca2+-depleted PSII. The uphill electron transfer pathway was induced by the significant increase in Em(S2/S3) caused by the loss of charge compensation for D2-Lys317 upon the depletion of Cl-, whereas it was induced by the significant decrease in Em(TyrZ) caused by the rearrangement of the water molecules at the Ca2+ binding moiety upon the depletion of Ca2+.

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina.

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c6 ) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A0 , A1 , and three Fe4 S4 clusters, FX , FA , and FB . Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A0 is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.

Lift the mystery veil on chlorophyllases

Photosystem II (PSII) is a large membrane-protein complex that catalyzes the splitting of water into protons, electrons, and oxygen via harvesting light energy (Johnson, 2016). The reaction center of PSII, operating like an electron pump, is a special pair of chlorophyll a (Chl a), referred to as P680, bound to D1 and D2 subunits, each with its set of electron carriers. Upon absorption of light energy, P680 is excited and donates an electron to the neighboring pheophytin (Pheo), producing a pair of radicals (⋅P680+ and ⋅Pheo−), called charge separation.

Nature of Asymmetric Electron Transfer in the Symmetric Pathways of Photosystem I.

Photosystem I has two active electron-transfer pathways. However, electron transfer occurs primarily along one of the two branches (A-branch) irrespective of the similar protein environments. Here, we report the origin of the A-branch electron transfer, considering the electronic coupling of the pigments and the electrostatic interaction with the protein environments. In the chlorophyll pair [PAPB], the electronic coupling between PA and PB is large (85 meV) for the highest occupied molecular orbital, forming the electronically coupled dimer [PAPB] and serving as an initial electron donor. In contrast, the coupling for the lowest unoccupied molecular orbital is small (15 meV), leading to charge transfer from PB to PA upon the [PAPB] excitation. The electronic coupling between [PAPB] and the accessory chlorophyll in the A-branch is significantly larger than that in the B-branch. These results indicate that the asymmetry of the electron-transfer activity originates from PA as a chlorophyll epimer.

Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210-1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg2+ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn4CaO5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1+PheoD1− radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of 1O2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to ChlD1 upon QA− formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 itself is the chlorophyll that is first damaged by 1O2, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2A/ec3A or ec2B/ec3B) and a phylloquinone (PhQA or PhQB). The axial ligand to the central Mg2+ of the ec2A and ec2B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to FX electron transfer show that in both species, the PsaA-N604L mutation (near ec2B) results in a ~50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2A) results in a ~70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P700+PhQ− is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.

Impact of molecular flexibility on the site energy shift of chlorophylls in Photosystem II

Light harvesting from the Sun by antenna complexes surrounding the reaction center of Photosystem II represents the first step of the natural oxygenic photosynthesis performed by plants, algae and cyanobacteria. The excitation energy derived from the sunlight is absorbed by the chlorophylls of the antenna and subsequently conveyed to the reaction center of Photosystem II through resonant energy transfer mechanisms. In the special pair of chlorophylls of the reaction center the charge separation occurs, eventually leading to the oxidation of water molecules into protons, electrons and molecular oxygen. The adsorption properties of the antenna chlorophylls are ad hoc modulated by the protein environment to guarantee fast energy transfer and minimize side and back reactions. At the same time these properties are influenced by the molecular fluctuations occurring at physiological temperature. In the present work, combining classical molecular dynamics simulations with the Charge Density Coupling method, we estimated the impact of the thermal fluctuations on the site energy shift of the chlorophylls embedded in the Photosystem II complex. Our results show how the effect of the molecular fluctuations is not homogeneous throughout the complex, although the symmetry of the homodimer is maintained. Many peripheral chromophores undergo fluctuations larger then 10kJ/mol around the average values. Possible physiological roles of such fluctuations are discussed.

Interaction of various types of photosystem I complexes with exogenous electron acceptors.

Interaction of photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 containing various quinones in the A1-site (phylloquinone PhQ in the wild-type strain (WT), and plastoquinone PQ or 2,3-dichloronaphthoquinone Cl 2 NQ in the menB deletion strain) and different numbers of Fe4S4 clusters (intact WT and FX-core complexes depleted of FA/FB centers) with external acceptors has been studied. The efficiency of interaction was estimated by measuring the light-induced absorption changes at 820nm due to the reduction of the special pair of chlorophylls (P700+) by an external acceptor(s). It was shown that externally added Cl 2 NQ is able to effectively accept electrons from the terminal iron-sulfur clusters of PS I. Moreover, the efficiency of Cl 2 NQ as external acceptor was higher than the efficiency of the commonly used artificial electron acceptor, methylviologen (MV) for both the intact WT PS I and for the FX-core complexes. The comparison of the efficiency of MV interaction with different types of PS I complexes revealed gradual decrease in the following order: intact WT > menB > FX-core. The effect of MV on the recombination kinetics in menB complexes of PS I with Cl 2 NQ in the A1-site differed significantly from all other PS I samples. The obtained effects are considered in terms of kinetic efficiency of electron acceptors in relation to thermodynamic and structural characteristics of PS I complexes.

Dynamics of the Special Pair of Chlorophylls of Photosystem II.

Cholophylls are at the basis of the photosynthetic energy conversion mechanisms in algae, plants, and cyanobacteria. In photosystem II, the photoproduced electrons leave a special pair of chlorophylls (namely, P(D1) and P(D2)) that becomes cationic. This oxidizing pair [P(D1),P(D2)](+), in turn, triggers a cascade of oxidative events, eventually leading to water splitting and oxygen evolution. In the present work, using quantum mechanics/molecular mechanics calculations, we investigate the electronic structure and the dynamics of the P(D1)P(D2) special pair in both its oxidized and reduced states. In agreement with previously reported static calculations, the symmetry between the two chlorophylls was found to be broken, the positive charge being preferentially located on P(D1). Nevertheless, this study reveals for the first time that large charge fluctuations occur along dynamics, temporarily inverting the charge preference for the two branches. Finally, a vibrational analysis pinpointed that such charge fluctuations are strongly coupled to specific modes of the special pair.

From light-harvesting to photoprotection: structural basis of the dynamic switch of the major antenna complex of plants (LHCII).

Light-Harvesting Complex II (LHCII) is largely responsible for light absorption and excitation energy transfer in plants in light-limiting conditions, while in high-light it participates in photoprotection. It is generally believed that LHCII can change its function by switching between different conformations. However, the underlying molecular picture has not been elucidated yet. The available crystal structures represent the quenched form of the complex, while solubilized LHCII has the properties of the unquenched state. To determine the structural changes involved in the switch and to identify potential quenching sites, we have explored the structural dynamics of LHCII, by performing a series of microsecond Molecular Dynamics simulations. We show that LHCII in the membrane differs substantially from the crystal and has the signatures that were experimentally associated with the light-harvesting state. Local conformational changes at the N-terminus and at the xanthophyll neoxanthin are found to strongly correlate with changes in the interactions energies of two putative quenching sites. In particular conformational disorder is observed at the terminal emitter resulting in large variations of the excitonic coupling strength of this chlorophyll pair. Our results strongly support the hypothesis that light-harvesting regulation in LHCII is coupled with structural changes.

Synthesis and Self-Organization of Zinc β-(Dialkoxyphosphoryl)porphyrins in the Solid State and in Solution

The first synthesis and self-organization of zinc β-phosphorylporphyrins in the solid state and in solution are reported. β-Dialkoxyphosphoryl-5,10,15,20-tetraphenylporphyrins and their Zn(II) complexes have been synthesized in good yields by using Pd- and Cu-mediated carbon-phosphorous bond-forming reactions. The Cu-mediated reaction allowed to prepare the mono-β-(dialkoxyphosphoryl)porphyrins 1 Zn-3 Zn starting from the β-bromo-substituted zinc porphyrinate ZnTPPBr (TPP = tetraphenylporphyrin) and dialkyl phosphites HP(O)(OR)(2) (R = Et, iPr, nBu). The derivatives 1 Zn-3 Zn were obtained in good yields by using one to three equivalents of CuI. When the reaction was carried out in the presence of catalytic amounts of palladium complexes in toluene, the desired zinc derivative 1 Zn was obtained in up to 72% yield. The use of a Pd-catalyzed C-P bond-forming reaction was further extended to the synthesis of β-poly(dialkoxyphosphoryl)porphyrins. An unprecedented one-pot sequence involving consecutive reduction and phosphorylation of H(2)TPPBr(4) led to the formation of a mixture of the 2,12- and 2,13-bis(dialkoxy)phosphorylporphyrins 5 H(2) and 6 H(2) in 81% total yield. According to the X-ray diffraction studies, 1 Zn and 3 Zn are partially overlapped cofacial dimers formed through the coordination of two Zn centers by two phosphoryl groups belonging to the adjacent molecules. The equilibrium between the monomeric and the dimeric species exists in solutions of 1 Zn and 3 Zn in weakly polar solvents according to spectroscopic data (UV/Vis absorption and NMR spectroscopy). The ratio of each form is dependent on the concentration, temperature, and traces of water or methanol. These features demonstrated that zinc β-phosphorylporphyrins can be regarded as new model compounds for the weakly coupled chlorophyll pair in the photosynthesis process.

Distribution of the Cationic State over the Chlorophyll Pair of the Photosystem II Reaction Center

The reaction center chlorophylls a (Chla) of photosystem II (PSII) are composed of six Chla molecules including the special pair Chla P(D1)/P(D2) harbored by the D1/D2 heterodimer. They serve as the ultimate electron abstractors for water oxidation in the oxygen-evolving Mn(4)CaO(5) cluster. Using the PSII crystal structure analyzed at 1.9 Å resolution, the redox potentials of P(D1)/P(D2) for one-electron oxidation (E(m)) were calculated by considering all PSII subunits and the protonation pattern of all titratable residues. The E(m)(Chla) values were calculated to be 1015-1132 mV for P(D1) and 1141-1201 mV for P(D2), depending on the protonation state of the Mn(4)CaO(5) cluster. The results showed that E(m)(P(D1)) was lower than E(m)(P(D2)), favoring localization of the charge of the cationic state more on P(D1). The P(D1)(•+)/P(D2)(•+) charge ratio determined by the large-scale QM/MM calculations with the explicit PSII protein environment yielded a P(D1)(•+)/P(D2)(•+) ratio of ~80/~20, which was found to be due to the asymmetry in electrostatic characters of several conserved D1/D2 residue pairs that cause the E(m)(P(D1))/E(m)(P(D2)) difference, e.g., D1-Asn181/D2-Arg180, D1-Asn298/D2-Arg294, D1-Asp61/D2-His61, D1-Glu189/D2-Phe188, and D1-Asp170/D2-Phe169. The larger P(D1)(•+) population than P(D2)(•+) appears to be an inevitable fate of the intact PSII that possesses water oxidation activity.

Independent initiation of primary electron transfer in the two branches of the photosystem I reaction center

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with approximately 100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P(700) (a pair of chlorophylls), and the tertiary acceptor, F(X) (a Fe(4)S(4) cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F(X), it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13(1)-keto oxygen of either ec3(A) (PsaA-Tyr696) or ec3(B) (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2(+)ec3(-) radical pair, followed by rapid reduction by P(700) in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.