Charge separation in the photosystem II reaction center resolved by multispectral two-dimensional electronic spectroscopy.

The photosystem II reaction center (PSII RC) performs the primary energy conversion steps of oxygenic photosynthesis. While the PSII RC has been studied extensively, the similar time scales of energy transfer and charge separation and the severely overlapping pigment transitions in the Q region have led to multiple models of its charge separation mechanism and excitonic structure. Here, we combine two-dimensional electronic spectroscopy (2DES) with a continuum probe and two-dimensional electronic vibrational spectroscopy (2DEV) to study the cyt b559-D1D2 PSII RC at 77 K.

Photosystem 2 and the oxygen evolving complex: a brief overview.

These special issues of photosynthesis research present papers documenting progress in revealing the many aspects of photosystem 2, a unique, one-of-a-kind complex system that can reduce a plastoquinone to a plastoquinol on every second flash of light and oxidize 2 HO to an O on every fourth flash. This overview is a brief personal assessment of the progress observed by the author over a four-decade research career, including a discussion of some remaining unsolved issues. It will come as no surprise to readers that there are remaining questions given the complexity of PS2, and the efforts that have been needed so far to uncover its secrets.

Vibronic coherence in oxygenic photosynthesis.

Photosynthesis powers life on our planet. The basic photosynthetic architecture consists of antenna complexes that harvest solar energy and reaction centres that convert the energy into stable separated charge. In oxygenic photosynthesis, the initial charge separation occurs in the photosystem II reaction centre, the only known natural enzyme that uses solar energy to split water.

Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II.

The N-terminal ¹E-⁶L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the ¹E-⁷T or ¹E-¹⁵T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII.

pH optimum of the photosystem II H₂O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O₂ evolution activity.

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim.

Probing the topography of the photosystem II oxygen evolving complex: PsbO is required for efficient calcium protection of the manganese cluster against dark-inhibition by an artificial reductant.

The photosystem II (PSII) manganese-stabilizing protein (PsbO) is known to be the essential PSII extrinsic subunit for stabilization and retention of the Mn and Cl(-) cofactors in the oxygen evolving complex (OEC) of PSII, but its function relative to Ca(2+) is less clear. To obtain a better insight into the relationship, if any, between PsbO and Ca(2+) binding in the OEC, samples with altered PsbO-PSII binding properties were probed for their potential to promote the ability of Ca(2+) to protect the Mn cluster against dark-inhibition by an exogenous artificial reductant, N,N-dimethylhydroxylamine. In the absence of the PsbP and PsbQ extrinsic subunits, Ca(2+) and its surrogates (Sr(2+), Cd(2+)) shield Mn atoms from inhibitory reduction (Kuntzleman et al.

Binding stoichiometry and affinity of the manganese-stabilizing protein affects redox reactions on the oxidizing side of photosystem II.

It has been reported previously that the two subunits of PsbO, the photosystem II (PSII) manganese stabilizing protein, have unique functions in relation to the Mn, Ca(2+), and Cl(-) cofactors in eukaryotic PSII [Popelkova; (2008) Biochemistry 47, 12593]. The experiments reported here utilize a set of N-terminal truncation mutants of PsbO, which exhibit altered subunit binding to PSII, to further characterize its role in establishing efficient O(2) evolution activity. The effects of PsbO binding stoichiometry, affinity, and specificity on Q(A)(-) reoxidation kinetics after a single turnover flash, S-state transitions, and O(2) release time have been examined.

PsbO, the manganese-stabilizing protein: analysis of the structure-function relations that provide insights into its role in photosystem II.

The minireview presented here summarizes current information on the structure and function of PsbO, the photosystem II (PSII) manganese-stabilizing protein, with an emphasis on the protein's assembly into PSII, and its function in facilitating rapid turnovers of the oxygen evolving reaction. Two putative mechanisms for functional assembly of PsbO, which behaves as an intrinsically disordered polypeptide in solution, into PSII are proposed. Finally, a model is presented for the role of PsbO in relation to the function of the Mn, Ca(2+), and Cl(-) cofactors that are required for water oxidation, as well as for the action of hydroxide and small Mn reductants that inhibit the function of the active site of the oxygen-evolving complex.

The double mutation ΔL6MW241F in PsbO, the photosystem II manganese stabilizing protein, yields insights into the evolution of its structure and function.

The W241F mutation in spinach manganese-stabilizing protein (PsbO) decreases binding to photosystem II (PSII); its thermostability is increased and reconstituted activity is lower [Wyman et al. (2008) Biochemistry 47, 6490-6498]. The results reported here show that W241F cannot adopt a normal solution structure and fails to reconstitute efficient Cl(-) retention by PSII.

Function of PsbO, the photosystem II manganese-stabilizing protein: probing the role of aspartic acid 157.

The D157N, D157E, and D157K mutations in the psbO gene encoding the photosystem II (PSII) manganese-stabilizing protein from spinach, exhibit near-wild-type PSII binding but are significantly impaired in O(2) evolution activity and Cl(-) retention by PSII [Popelkova et al. (2009) Biochemistry 48, 11920-11928]. To better characterize the role of PsbO-Asp157 in eukaryotic PSII, the effect of mutations in Asp157 on heat-induced changes in PsbO solution structure, O(2) release kinetics, and PSII redox reactions both within and outside the oxygen-evolving complex (OEC) have been examined.

Asp157 is required for the function of PsbO, the photosystem II manganese stabilizing protein.

PsbO, the photosystem II manganese stabilizing protein, contains an aspartate residue [Asp157 (spinach numbering)], which is highly conserved in eukaryotic and prokaryotic PsbOs. The homology model of the PSII-bound conformation of spinach PsbO presented here positions Asp157 in the large flexible loop of the protein. We have characterized site-directed mutants (D157N, D157E, and D157K) of spinach PsbO that were rebound to PsbO-depleted PSII to probe the role of Asp157.

Inorganic cofactor stabilization and retention: the unique functions of the two PsbO subunits of eukaryotic photosystem II.

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H.

S-state dependence of the calcium requirement and binding characteristics in the oxygen-evolving complex of photosystem II.

The functional role of the Ca (2+) ion in the oxygen-evolving complex of photosystem II is not yet clear. Current models explain why the redox cycle of the complex would be interrupted after the S 3 state without Ca (2+), but the literature shows that it is interrupted after the S 2 state. Reinterpretation of the literature on methods of Ca (2+) depletion [Miqyass, M.

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties.

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein.

Current status of the role of Cl(-) ion in the oxygen-evolving complex.

This minireview summarizes the current state of knowledge concerning the role of Cl(-) in the oxygen-evolving complex (OEC) of photosystem II (PSII). The model that proposes that Cl(-) is a Mn ligand is discussed in light of more recent work. Studies of Cl(-) specificity, stoichiometry, kinetics, and retention by extrinsic polypeptides are discussed, as are the results that fail to detect Cl(-) ligation to Mn and results that show a lack of a requirement for Cl(-) in PSII-catalyzed H(2)O oxidation.

Structure and function of photosystems I and II.

Oxygenic photosynthesis, the principal converter of sunlight into chemical energy on earth, is catalyzed by four multi-subunit membrane-protein complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b(6)f complex, and F-ATPase. PSI generates the most negative redox potential in nature and largely determines the global amount of enthalpy in living systems. PSII generates an oxidant whose redox potential is high enough to enable it to oxidize H(2)O, a substrate so abundant that it assures a practically unlimited electron source for life on earth.

Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.

Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Arg151 and Arg161 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced.

Amino acid sequences and solution structures of manganese stabilizing protein that affect reconstitution of Photosystem II activity.

This minireview presents a summary of information available on the secondary and tertiary structure of manganese stabilizing protein (MSP) in solution, and on the identity of amino acid residues that affect binding and functional assembly of this protein into Photosystem II. New data on the secondary structure of C-terminal mutants and 90 degrees C-heated manganese stabilizing protein, along with earlier data on the secondary structure of N-terminal mutants and the tertiary structure of all modified MSP species, allow for an evaluation of models for spinach MSP secondary and tertiary structure. This summary of previous and new information better documents the natively unfolded behavior of the protein in solution.

Structure and activity of the photosystem II manganese-stabilizing protein: role of the conserved disulfide bond.

The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme's active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution.

Assembly and function of the photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior.

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404-414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S-S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al.

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme.

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex.

Reconstitution of the photosystem II Ca2+ binding site.

The roles of Ca(2+) in H(2)O oxidation may be as a site of substrate binding, and as a structural component of the photosystem II O(2)-evolving complex. One indication of this dual role of the metal is revealed by probing the Mn cluster in the Ca(2+) depleted O(2) evolving complex that retains extrinsic 23- and 17-kDa polypeptides with reductants (NH(2)OH and hydroquinone) [Biochemistry 41 (2002) 958]. Calcium appears to bind to photosystem II at a site where it could bind substrate H(2)O.

Binding of manganese stabilizing protein to photosystem II: identification of essential N-terminal threonine residues and domains that prevent nonspecific binding.

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, (4)KRLTYD(10)E and (15)TYL(18)E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized.