Bridgette A. Barry

Proton coupled electron transfer and redox active tyrosines in Photosystem II

In this article, progress in understanding proton coupled electron transfer (PCET) in Photosystem II is reviewed. Changes in acidity/basicity may accompany oxidation/reduction reactions in biological catalysis. Alterations in the proton transfer pathway can then be used to alter the rates of the electron transfer reactions. Studies of the bioenergetic complexes have played a central role in advancing our understanding of PCET. Because oxidation of the tyrosine results in deprotonation of the phenolic oxygen, redox active tyrosines are involved in PCET reactions in several enzymes. This review focuses on PCET involving the redox active tyrosines in Photosystem II. Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone. Photosystem II provides a paradigm for the study of redox active tyrosines, because this photosynthetic reaction center contains two tyrosines with different roles in catalysis. The tyrosines, YZ and YD, exhibit differences in kinetics and midpoint potentials, and these differences may be due to noncovalent interactions with the protein environment. Here, studies of YD and YZ and relevant model compounds are described.

Posttranslational modifications in the CP43 subunit of photosystem II

Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123–133]. The sequence A350PWLEPLR357 in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.

Proton-coupled electron transfer in photosystem II: proton inventory of a redox active tyrosine.

Photosystem II (PSII) catalyzes the light driven oxidation of water and the reduction of plastoquinone. PSII is a multisubunit membrane protein; the D1 and D2 polypeptides form the heterodimeric core of the PSII complex. Water oxidation occurs at a manganese-containing oxygen evolving complex (OEC). PSII contains two redox active tyrosines, Y(Z) and Y(D), which form the neutral tyrosyl radicals, Y(z)(*) and Y(D)(*). Y(D) has been assigned as tyrosine 160 in the D2 polypeptide through isotopic labeling and site-directed mutagenesis. Whereas Y(D) is not directly involved in the oxidation of water, it has been implicated in the formation and stabilization of the OEC. PSII structures have shown Y(D) to be within hydrogen-bonding distance of histidine 189 in the D2 polypeptide. Spectroscopic studies have suggested that a proton is transferred between Y(D) and histidine 189 when Y(D) is oxidized and reduced. In our previous work, we used (2)H(2)O solvent exchange to demonstrate that the mechanism of Y(D) proton-coupled electron transfer (PCET) differs at high and low pH. In this article, we utilize the proton inventory technique to obtain more information concerning PCET mechanism at high pH. The hypercurvature of the proton inventory data provides evidence for the existence of multiple, proton-donation pathways to Y(D)(*). In addition, at least one of these pathways must involve the transfer of more than one proton.

THE ROLE OF REDOX‐ACTIVE AMINO ACIDS IN THE PHOTOSYNTHETIC WATER‐OXIDIZING COMPLEX

The protein environment can dramatically affect the EPR line shape of tyrosine radicals. The alterations can be caused by: (1) a change in methylene geometry caused by different protein steric constraints; (2) a change in spin density caused by a change in protein environment; or (3) covalent modification of the tyrosine. Any or all of these effects may also be important, in some cases, in control of oxidation potential and chemical reactivity. The new signal that has been observed in the YF161D1 PS II mutant has an approximate 1:3:3:1 lineshape. There is no precedent for a 1:3:3:1 EPR signal from a tyrosine in a powder sample. However, as described above, given the diversity of signals from tyrosine radicals, it is impossible to exclude the possibility that the signal arises from tyrosine on this basis.

The Use of Cyanobacteria in the Study of the Structure and Function of Photosystem II

Oxygenic photosynthesis occurs in plants, green algae, and procaryotic cyanobacteria. Two chlorophyll-containing photosystems cooperate to transfer electrons from water to NADP+. Photosystem II is the membrane protein complex that carries out the light-catalyzed oxidation of water and reduction of plastoquinone. The reaction center is composed of both intrinsic and extrinsic proteins; the prosthetic groups involved in electron transfer include chlorophyll, pheophytin, quinone, tyrosine residues, and a manganese cluster. Cyanobacteria have emerged as a convenient system with which to study the structure and function of Photosystem II for two reasons. Firstly, isotopic labeling experiments are possible in this organism, facilitating many types of biophysical experiments. Secondly, site-directed mutagenesis is easily performed. This chapter will review what is known about the structure and function of Photosystem II with particular emphasis on the use of cyanobacteria in such studies. Areas in which there are significant differences between plants and cyanobacteria will be highlighted.

A hydrogen-bonding network plays a catalytic role in photosynthetic oxygen evolution

In photosystem II, oxygen evolution occurs by the accumulation of photo-induced oxidizing equivalents at the oxygen-evolving complex (OEC). The sequentially oxidized states are called the S0-S4 states, and the dark stable state is S1. Hydrogen bonds to water form a network around the OEC; this network is predicted to involve multiple peptide carbonyl groups. In this work, we tested the idea that a network of hydrogen bonded water molecules plays a catalytic role in water oxidation. As probes, we used OEC peptide carbonyl frequencies, the substrate-based inhibitor, ammonia, and the sugar, trehalose. Reaction-induced FT-IR spectroscopy was used to describe the protein dynamics associated with the S1 to S2 transition. A shift in an amide CO vibrational frequency (1664 (S1) to 1653 (S2) cm-1) was observed, consistent with an increase in hydrogen bond strength when the OEC is oxidized. Treatment with ammonia/ammonium altered these CO vibrational frequencies. The ammonia-induced spectral changes are attributed to alterations in hydrogen bonding, when ammonia/ammonium is incorporated into the OEC hydrogen bond network. The ammonia-induced changes in CO frequency were reversed or blocked when trehalose was substituted for sucrose. This trehalose effect is attributed to a displacement of ammonia molecules from the hydrogen bond network. These results imply that ammonia, and by extension water, participate in a catalytically essential hydrogen bond network, which involves OEC peptide CO groups. Comparison to the ammonia transporter, AmtB, reveals structural similarities with the bound water network in the OEC.

Proton Coupled Electron Transfer and Redox-Active Tyrosine Z in the Photosynthetic Oxygen-Evolving Complex

Proton coupled electron transfer (PCET) reactions play an essential role in many enzymatic processes. In PCET, redox-active tyrosines may be involved as intermediates when the oxidized phenolic side chain deprotonates. Photosystem II (PSII) is an excellent framework for studying PCET reactions, because it contains two redox-active tyrosines, YD and YZ, with different roles in catalysis. One of the redox-active tyrosines, YZ, is essential for oxygen evolution and is rapidly reduced by the manganese-catalytic site. In this report, we investigate the mechanism of YZ PCET in oxygen-evolving PSII. To isolate YZ(•) reactions, but retain the manganese-calcium cluster, low temperatures were used to block the oxidation of the metal cluster, high microwave powers were used to saturate the YD(•) EPR signal, and YZ(•) decay kinetics were measured with EPR spectroscopy. Analysis of the pH and solvent isotope dependence was performed. The rate of YZ(•) decay exhibited a significant solvent isotope effect, and the rate of recombination and the solvent isotope effect were pH independent from pH 5.0 to 7.5. These results are consistent with a rate-limiting, coupled proton electron transfer (CPET) reaction and are contrasted to results obtained for YD(•) decay kinetics at low pH. This effect may be mediated by an extensive hydrogen-bond network around YZ. These experiments imply that PCET reactions distinguish the two PSII redox-active tyrosines.

N-formylkynurenine as a marker of high light stress in photosynthesis.

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ± 0.5) under high light illumination. A concomitant 2.4 ± 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.

DEPROTONATION OF THE 33-KDA, EXTRINSIC, MANGANESE-STABILIZING SUBUNIT ACCOMPANIES PHOTOOXIDATION OF MANGANESE IN PHOTOSYSTEM II

Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform 13C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S1 to S2transition in the presence either of reconstituted 12C or13C MSP. 13C labeling of MSP is shown to cause 30–60 cm−1 shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/aspartic acid residues on MSP during the S1 to S2 transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.

Detection of an intermediary, protonated water cluster in photosynthetic oxygen evolution.

In photosynthesis, photosystem II evolves oxygen from water by the accumulation of photooxidizing equivalents at the oxygen-evolving complex (OEC). The OEC is a Mn4CaO5 cluster, and its sequentially oxidized states are termed the Sn states. The dark-stable state is S1, and oxygen is released during the transition from S3 to S0. In this study, a laser flash induces the S1 to S2 transition, which corresponds to the oxidation of Mn(III) to Mn(IV). A broad infrared band, at 2,880 cm−1, is produced during this transition. Experiments using ammonia and 2H2O assign this band to a cationic cluster of internal water molecules, termed “W5+.” Observation of the W5+ band is dependent on the presence of calcium, and flash dependence is observed. These data provide evidence that manganese oxidation during the S1 to S2 transition results in a coupled proton transfer to a substrate-containing, internal water cluster in the OEC hydrogen-bonded network.