Redox-active Tyrosine Research Articles

Electrochemistry-enabled residue-specific modification of peptides and proteins.

Selective chemical reactions at precise amino acid residues of peptides and proteins have become an exploding field of research in the last few decades. With the emerging utility of bioconjugated peptides and proteins as drug leads and therapeutic agents, the design of smart protocols to modulate and conjugate biomolecules has become necessary. During this modification, the most important concern of biochemists is to keep intact the structural integrity of the biomolecules. Hence, a soft and selective biocompatible reaction environment is necessary. Electrochemistry, a mild and elegant tunable reaction platform to synthesize complex molecules while avoiding harsh and toxic chemicals, can provide such a reaction condition. However, this strategy is yet to be fully exploited in the field of selective modification of polypeptides. With this possibility, the use of electrochemistry as a reaction toolbox in peptide and protein chemistry is flourishing day by day. Unfortunately, there is no suitable review article summarizing the residue-specific modification of biomolecules. The present review provides a comprehensive summary of the latest manifested electrochemical approaches for the modulation of five redox-active amino acid residues, namely cysteine, tyrosine, tryptophan, histidine and methionine, found in peptides and proteins. The article also highlights the incredible potential of electrochemistry for the regio- as well as chemoselective bioconjugation strategy of biomolecules.

Tracking the first electron transfer step at the donor side of oxygen-evolving photosystem II by time-resolved infrared spectroscopy.

In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680+) precedes the water-oxidation chemistry of the S-state cycle of the Mn4Ca cluster. Here we investigate these early events, observable within about 10ns to 10ms after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310-1890cm-1 for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80ns, 500ns, and 10µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680+ was used to trace largely specifically the P680+ reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680+ transients with a sum of four exponentials, revealing two nanosecond phases (30-46ns and 690-1110ns) and two microsecond phases (4.5-8.3µs and 42µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry.

Role of Secondary Coordination Sphere Residues in Halogenation Catalysis of Non-heme Iron Enzymes

Chemo- and regio-selective catalysis of the C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-Oxoglutarate (2OG)-dependent non-heme iron halogenases catalyze selective chlorination/bromination of C–H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well understood. In this work, we show that specific positioning of redox-active tyrosine residues in the SCS of non-heme iron halogenases has a huge impact on their structure, function, and reactivity. We discover that a tyrosine residue (F121Y) rationally incorporated to hydrogen bond to iron’s chloride ligand in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies demonstrate that DOPA modification in SyrB2 renders the enzyme non-functional. Bioinformatic analysis suggests that SyrB2-like halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Furthermore, molecular dynamics simulations in tandem with experimental demonstration of DOPA incorporation exclusively at position 121 enables us to uniquely identify that an axial-chloro haloferryl isomer is operant in SyrB2. We also identify conserved redox-inactive residues in the SCS of other 2OG-dependent non-heme iron halogenases to avoid DOPA-like unproductive oxidations. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases and will have significant implications toward the design of small-molecule and protein-based halogenation catalysts.

Insights into the Thermodynamics and Kinetics of Amino-Acid Radicals in Proteins.

Some oxidoreductase enzymes use redox-active tyrosine, tryptophan, cysteine, and/or glycine residues as one-electron, high-potential redox (radical) cofactors. Amino-acid radical cofactors typically perform one of four tasks-they work in concert with a metallocofactor to carry out a multielectron redox process, serve as storage sites for oxidizing equivalents, activate the substrate molecules, or move oxidizing equivalents over long distances. It is challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single-amino-acid residue. The inherently reactive and highly oxidizing properties of amino-acid radicals increase the experimental barriers further still. This review describes a family of stable and well-structured model proteins that was made specifically to study tyrosine and tryptophan oxidation-reduction. The so-called α3X model protein system was combined with very-high-potential protein film voltammetry, transient absorption spectroscopy, and theoretical methods to gain a comprehensive description of the thermodynamic and kinetic properties of protein tyrosine and tryptophan radicals.

The Redox-Active Tyrosine Is Essential for Proton Pumping in Cytochrome c Oxidase

Cellular respiration involves electron transport via a number of enzyme complexes to the terminal Cytochrome c oxidase (CcO), in which molecular oxygen is reduced to water. The free energy released in the reduction process is used to establish a transmembrane electrochemical gradient, via two processes, both corresponding to charge transport across the membrane in which the enzymes are embedded. First, the reduction chemistry occurring in the active site of CcO is electrogenic, which means that the electrons and protons are delivered from opposite sides of the membrane. Second, the exergonic chemistry is coupled to translocation of protons across the entire membrane, referred to as proton pumping. In the largest subfamily of the CcO enzymes, the A-family, one proton is pumped for every electron needed for the chemistry, making the energy conservation particularly efficient. In the present study, hybrid density functional calculations are performed on a model of the A-family CcOs. The calculations show that the redox-active tyrosine, conserved in all types of CcOs, plays an essential role for the energy conservation. Based on the calculations a reaction mechanism is suggested involving a tyrosyl radical (possibly mixed with tyrosinate character) in all reduction steps. The result is that the free energy released in each reduction step is large enough to allow proton pumping in all reduction steps without prohibitively high barriers when the gradient is present. Furthermore, the unprotonated tyrosine provides a mechanism for coupling the uptake of two protons per electron in every reduction step, i.e. for a secure proton pumping.

Trehalose matrix effects on electron transfer in Mn-depleted protein-pigment complexes of Photosystem II

The kinetics of flash-induced re-reduction of the Photosystem II (PS II) primary electron donor P680 was studied in solution and in trehalose glassy matrices at different relative humidity. In solution, and in the re-dissolved glass, kinetics were dominated by two fast components with lifetimes in the range of 2–7 μs, which accounted for >85% of the decay. These components were ascribed to the direct electron transfer from the redox-active tyrosine YZ to P680+. The minor slower components were due to charge recombination between the primary plastoquinone acceptor QA− and P680+. Incorporation of the PS II complex into the trehalose glassy matrix and its successive dehydration caused a progressive increase in the lifetime of all kinetic phases, accompanied by an increase of the amplitudes of the slower phases at the expense of the faster phases. At 63% relative humidity the fast components contribution dropped to ~50%. A further dehydration of the trehalose glass did not change the lifetimes and contribution of the kinetic components. This effect was ascribed to the decrease of conformational mobility of the protein domain between YZ and P680, which resulted in the inhibition of YZ → P680+ electron transfer in about half of the PS II population, wherein the recombination between QA− and P680+ occurred. The data indicate that PS II binds a larger number of water molecules as compared to PS I complexes. We conclude that our data disprove the “water replacement” hypothesis of trehalose matrix biopreservation.

Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese‑calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

HYSCORE and DFT Studies of Proton-Coupled Electron Transfer in a Bioinspired Artificial Photosynthetic Reaction Center.

SummaryThe photosynthetic water-oxidation reaction is catalyzed by the oxygen-evolving complex in photosystem II (PSII) that comprises the Mn4CaO5 cluster, with participation of the redox-active tyrosine residue (YZ) and a hydrogen-bonded network of amino acids and water molecules. It has been proposed that the strong hydrogen bond between YZ and D1-His190 likely renders YZ kinetically and thermodynamically competent leading to highly efficient water oxidation. However, a detailed understanding of the proton-coupled electron transfer (PCET) at YZ remains elusive owing to the transient nature of its intermediate states involving YZ⋅. Herein, we employ a combination of high-resolution two-dimensional 14N hyperfine sublevel correlation spectroscopy and density functional theory methods to investigate a bioinspired artificial photosynthetic reaction center that mimics the PCET process involving the YZ residue of PSII. Our results underscore the importance of proximal water molecules and charge delocalization on the electronic structure of the artificial reaction center.

Energetics of Ionized Water Molecules in the H-Bond Network near the Ca2+ and Cl- Binding Sites in Photosystem II.

In photosystem II, water oxidation occurs at the oxygen-evolving complex (OEC). The presence of a hydronium ion (H3O+) was proposed at the Cl- binding site and Ca2+-depleted OEC. Using a quantum mechanical/molecular mechanical approach, we report the stability of H3O+ in the PSII protein environment. Neither release of the proton from ligand water molecule W2 at the OEC nor formation of H3O+ at Cl- is energetically favorable. In contrast, H3O+ can exist at the Ca2+-depleted OEC. Even when H3O+ exists in Ca2+-depleted PSII, the H-bond network of the redox-active tyrosine (TyrZ) remains unaltered, retaining the unusually short low-barrier H-bond with D1-His190, and the redox potential of TyrZ, Em(TyrZ), remains unaltered. These observations explain why the oxidation of the Ca2+-depleted Mn4O5 cluster by TyrZ (i.e., the S2 to S3 transition) is not inhibited at low pH. It seems likely that Ca2+ plays a role in not only (i) maintaining the H-bond network and facilitating TyrZ oxidation [tuning Em(TyrZ)] but also (ii) providing the valence of +2, decreasing the pKa of the ligand molecule (W1), and facilitating the release of the proton from W1 in the S2 to S3 transition together with Cl-.

Actinobacterial Coproheme Decarboxylases Use Histidine as a Distal Base to Promote Compound I Formation.

Coproheme decarboxylases (ChdCs) catalyze the final step in heme b biosynthesis of monoderm and some diderm bacteria. In this reaction, coproheme is converted to heme b via monovinyl monopropionate deuteroheme (MMD) in two consecutive decarboxylation steps. In Firmicutes decarboxylation of propionates 2 and 4 of coproheme depend on hydrogen peroxide and the presence of a catalytic tyrosine. Here we demonstrate that ChdCs from Actinobacteria are unique in using a histidine (H118 in ChdC from Corynebacterium diphtheriae, CdChdC) as a distal base in addition to the redox-active tyrosine (Y135). We present the X-ray crystal structures of coproheme-CdChdC and MMD-CdChdC, which clearly show (i) differences in the active site architecture between Firmicutes and Actinobacteria and (ii) rotation of the redox-active reaction intermediate (MMD) after formation of the vinyl group at position 2. Distal H118 is shown to catalyze the heterolytic cleavage of hydrogen peroxide (kapp = (4.90 ± 1.25) × 104 M–1 s–1). The resulting Compound I is rapidly converted to a catalytically active Compound I* (oxoiron(IV) Y135•) that initiates the radical decarboxylation reactions. As a consequence of the more efficient Compound I formation, actinobacterial ChdCs exhibit a higher catalytic efficiency in comparison to representatives from Firmicutes. On the basis of the kinetic data of wild-type CdChdC and the variants H118A, Y135A, and H118A/Y135A together with high-resolution crystal structures and molecular dynamics simulations, we present a molecular mechanism for the hydrogen peroxide dependent conversion of coproheme via MMD to heme b and discuss differences between ChdCs from Actinobacteria and Firmicutes.

Exploring Coupled Redox and pH Processes with a Force-Field-Based Approach: Applications to Five Different Systems.

Coupled redox and pH-driven processes are at the core of many important biological mechanisms. As the distribution of protonation and redox states in a system is associated with the pH and redox potential of the solution, having efficient computational tools that can simulate under these conditions becomes very important. Such tools have the potential to provide information that complement and drive experiments. In previous publications we have presented the implementation of the constant pH and redox potential molecular dynamics (C(pH,E)MD) method in AMBER and we have shown how multidimensional replica exchange can be used to significantly enhance the convergence efficiency of our simulations. In the current work, after an improvement in our C(pH,E)MD approach that allows a given residue to be simultaneously pH- and redox-active, we have employed our methodologies to study five different systems of interest in the literature. We present results for capped tyrosine dipeptide, two maquette systems containing one pH- and redox-active tyrosine (α3Y and peptide A), and two proteins that contain multiple heme groups (diheme cytochrome c from Rhodobacter sphaeroides and Desulfovibrio vulgaris Hildenborough cytochrome c3). We show that our results can provide new insights into previous theoretical and experimental findings by using a fully force-field-based and GPU-accelerated approach, which allows the simulations to be executed with high computational performance.

Pivotal role of the redox-active tyrosine in driving the water splitting catalyzed by photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4Ca cluster in photosystem II (PSII). The nearby redox-active tyrosine (YZ) serves as a direct electron acceptor of the Mn4Ca cluster and it forms a low-barrier H-bond (LBHB) with a neighboring histidine residue (D1-His190). Experimental evidence indicates that YZ oxidation triggers changes in the hydrogen bonding network that precede proton abstraction from the Mn4Ca cluster. In order to characterize such changes, we compare ab initio molecular dynamics simulations of different states of the catalytic cycle of PSII with dynamics of isolated tyrosine models (namely, p-cresol) in different oxidation states. The systematic comparison of the H-bond networks in different simulated systems suggests that the YZ oxidation leads to a water hydration pattern which is more similar to that of the neutral p-cresol rather than that of the p-cresol anion. Our simulations also reveal the twofold nature of the interactions between YZ and the Mn4Ca cluster. Firstly, the YZ oxidation triggers rapid structural changes of the H-bond pattern in the proximity of the cluster which have been observed to propagate on the ps time scale on the Ca2+ hydration shell up to other water molecules in the proximity of the cluster. Secondly, it is clear that YZ interacts with the Mn4Ca cluster also through Coulombic interactions mediated by CP43-Arg357 through the remaining positive charge of the pair. Our results are able to identify, for the first time, the structural rearrangements guided by the oxidation of YZ necessary for the evolution of the water splitting reaction in PSII. Based on these findings, we propose a mechanism of structural changes which is functional towards the progression of the catalytic cycle in PSII.

Redox Potential of the Oxygen-Evolving Complex in the Electron Transfer Cascade of Photosystem II.

In photosystem II (PSII), water oxidation occurs in the Mn4CaO5 cluster with the release of electrons via the redox-active tyrosine (TyrZ) to the reaction-center chlorophylls (PD1/PD2). Using a quantum mechanical/molecular mechanical approach, we report the redox potentials (Em) of these cofactors in the PSII protein environment. The Em values suggest that the Mn4CaO5 cluster, TyrZ, and PD1/PD2 form a downhill electron transfer pathway. Em for the first oxidation step, Em(S0/S1), is uniquely low (730 mV) and is ∼100 mV lower than that for the second oxidation step, Em(S1/S2) (830 mV) only when the O4 site of the Mn4CaO5 cluster is protonated in S0. The O4-water chain, which directly forms a low-barrier H-bond with the Mn4CaO5 cluster and mediates proton-coupled electron transfer in the S0 to S1 transition, explains why the second lowest oxidation state, S1, is the most stable and S0 is converted to S1 even in the dark.

Electron Transfer on the Donor Side of Manganese-Depleted Photosystem 2.

After removal of manganese ions responsible for light-driven water oxidation, redox-active tyrosine YZ (tyrosine 161 of the D1 subunit) still remains the dominant electron donor to the photooxidized chlorophyll P680 (P680+) in the reaction center of photosystem 2 (PS2). Here, we investigated P680+ reduction by YZ under single-turnover flashes in Mn-depleted PS2 core complexes in the presence of weak acids and NH4Cl. Analysis of changes in the light-induced absorption at 830 nm (reflecting P680 redox transitions) at pH 6.0 showed that P680+ reduction is well approximated by two kinetic components with the characteristic times (τ) of ~7 and ~31 μs and relative contributions of ~54 and ~37%, respectively. In contrast to the very small effect of sodium formate (200 mM), addition of sodium acetate and NH4Cl increased the rate of electron transfer between YZ and P680+ approx. by a factor of 5. The suggestion that direct electron transfer from YZ to P680+ has a biphasic kinetics and reflects the presence of two different populations of PS2 centers was confirmed by the data obtained using direct electrometrical technique. It was demonstrated that the submillisecond two-phase kinetics of the additional electrogenic phase in the kinetics of photoelectric response due to the electron transfer between YZ and P680+ is significantly accelerated in the presence of acetate or ammonia. These results contribute to the understanding of the mechanism of interaction between the oxidized tyrosine YZ and exogenous substances (including synthetic manganese-containing compounds) capable of photooxidation of water molecule in the manganese-depleted PS2 complexes.

Light sensitivity of the photoreceptor cryptochrome of the Drosophila circadian clock and its interaction with other clock components

Flavoprotein cryptochromes (CRYs) are blue light sensors that regulate the circadian clock in plants and animals. They consist of a highly conserved photolyase homology region (PHR) and a C‐terminal tail extension (CTE) of various sizes. In Drosophila CRY, photoreduction of the PHR bound cofactor FAD triggers conformational changes within CRY, which include undocking of C‐terminal Tail (CTT) helix. The mechanism of FAD photoreduction remains a key question in understanding CRY activation.Four tryptophan residues (W420, W397, W342, W394) mediate electron transfer for flavin photoreduction. We have substituted these residues to both redox‐inert phenylalanine residues (F) and redox‐active tyrosine (Y) residues to tune the light sensitivity of CRY. Light‐sensitivities of the variants correlate well with the extent of conformational change at the CTT. Interestingly, the surface tryptophan W394 is the most indispensable residue because it gates electron transfer from exogenous reductant to the photoreduction chain.In flies, CRY interacts with other signaling proteins of the circadian clock in a light depend‐manner to entrain the 24 hour‐cycle to environmental light. For example, in light CRY binds the protein timeless (TIM) and recruits the E3 ligase jetlag to facilitate TIM degradation. However, the molecular interactions between CRY and TIM are not well understood due to the low stability of this protein complex. We have utilized a chemical cross‐linking strategy to map the interfacing domain in this assembly and stabilize it for future structure study by structural methodsSupport or Funding InformationThis work was supported by grants from the NIH: R01GM054339 (to M.W.Y.) and R35GM122535 (to B.R.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proton Translocation via Tautomerization of Asn298 During the S2-S3 State Transition in the Oxygen-Evolving Complex of Photosystem II.

In biological water oxidation, a redox-active tyrosine residue (D1-Tyr161 or YZ) mediates electron transfer between the Mn4CaO5 cluster of the oxygen-evolving complex and the charge-separation site of photosystem II (PSII), driving the cluster through progressively higher oxidation states Si (i = 0–4). In contrast to lower S-states (S0, S1), in higher S-states (S2, S3) of the Mn4CaO5 cluster, YZ cannot be oxidized at cryogenic temperatures due to the accumulation of positive charge in the S1 → S2 transition. However, oxidation of YZ by illumination of S2 at 77–190 K followed by rapid freezing and charge recombination between YZ• and the plastoquinone radical QA•– allows trapping of an S2 variant, the so-called S2trapped state (S2t), that is capable of forming YZ• at cryogenic temperature. To identify the differences between the S2 and S2t states, we used the S2tYZ• intermediate as a probe for the S2t state and followed the S2tYZ•/QA•– recombination kinetics at 10 K using time-resolved electron paramagnetic resonance spectroscopy in H2O and D2O. The results show that while S2tYZ•/QA•– recombination can be described as pure electron transfer occurring in the Marcus inverted region, the S2t → S2 reversion depends on proton rearrangement and exhibits a strong kinetic isotope effect. This suggests that YZ oxidation in the S2t state is facilitated by favorable proton redistribution in the vicinity of YZ, most likely within the hydrogen-bonded YZ–His190–Asn298 triad. Computational models show that tautomerization of Asn298 to its imidic acid form enables proton translocation to an adjacent asparagine-rich cavity of water molecules that functions as a proton reservoir and can further participate in proton egress to the lumen.

Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product.

Environmental pH and a Glu364 to Gln mutation in the chlorophyll-binding CP47 protein affect redox-active TyrD and charge recombination in Photosystem II.

In Photosystem II, loop E of the chlorophyll-binding CP47 protein is located near a redox-active tyrosine, YD , forming a symmetrical analog to loop E in CP43, which provides a ligand to the oxygen-evolving complex (OEC). A Glu364 to Gln substitution in CP47, near YD , does not affect growth in the cyanobacterium Synechocystis sp. PCC 6803; however, deletion of the extrinsic protein PsbV in this mutant leads to a strain displaying a pH-sensitive phenotype. Using thermoluminescence, chlorophyll fluorescence, and flash-induced oxygen evolution analyses, we demonstrate that Glu364 influences the stability of YD and the redox state of the OEC, and highlight the effects of external pH on photosynthetic electron transfer in intact cyanobacterial cells.

Mechanism of Radical Formation in the H-Bond Network of D1-Asn298 in Photosystem II.

In photosystem II (PSII), redox-active tyrosine Z (TyrZ) forms a low-barrier H-bond with Nε of D1-His190. The PSII crystal structures show that Nδ of D1-His190 donates an H-bond to the carbonyl O of D1-Asn298. However, at a level of ∼2 Å resolution, a clear discrimination between the -NH2 and -C═O groups of the asparagine side chain may not be possible based on the electron density map. Using quantum mechanical/molecular mechanical calculations, we investigated the energetics of the D1-Asn298 conformations. In the D1-Asn298-rotated conformation, where the amide N group donates an H-bond to deprotonated Nδ of D1-His190, oxidation of S2 resulted in formation of a neutral radical, either TyrZ• or D1-His190•. This suggests that in the D1-Asn298-rotated conformation, the redox potential ( Em) values of TyrZ/D1-His190 are lower than the Em of the Mn4CaO5 cluster due to deprotonated D1-His190. The large disorder of a water molecule (water 1117A) at D1-Asn298 in the crystal structure as well as the absence of water 1117A in the Sr2+-substituted crystal structure may be associated with coexistence of the two D1-Asn298 conformations in the crystals.

Electrogenic reactions in Mn-depleted photosystem II core particles in the presence of synthetic binuclear Mn complexes

An electrometrical technique was used to investigate electron transfer between synthetic binuclear manganese (Mn) complexes, designated M − 2 and M − 3, and the redox-active neutral tyrosine radical (YZ•) in proteoliposomes containing Mn-depleted photosystem II (PS II) core particles in response to single laser flashes. In the absence of Mn-containing compounds, the observed flash-induced membrane potential (ΔΨ) decay was mainly due to charge recombination between the reduced primary quinone acceptor QA− and the oxidized YZ•. More significant slowing down of the ΔΨ decay in the presence of lower concentrations of M − 2 and M − 3 associated with electron donation from Mn in the Mn-binding site to YZ• indicates that these synthetic compounds are more effective electron donors than MnCl2. The exponential fitting of the kinetics of additional electrogenic components of ΔΨ rise in the presence of Mn-containing compounds revealed the following relative amplitudes (A) and lifetimes (τ): for MnCl2 - A∼ 3.5, τ∼150 μs, for M − 2 - A∼5%, τ∼1.4 ms, and for M − 3 - A∼5.5%, τ∼150 μs. This suggests that the efficiency of the manganese complexes in electron donation depends on the chemical nature of ligands. The experiments with EDTA-treated samples indicated that the ligands for M − 2 and M − 3 are required for their tight binding with the PS II reaction center. The obtained results demonstrate the importance of understanding the molecular mechanism(s) of flash-induced electrogenic reduction of the tyrosine radical YZ• by synthetic Mn complexes capable of splitting water into oxygen and reducing equivalents.