E. coli Ribonucleotide Reductase ß2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine-Fe(II) Adduct.

Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR ß2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR ß2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the ß2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (Kd ∼ 6 nM) of 3AP-Fe(II) for ß2. Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.

Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using Reaction-Induced FTIR Spectroscopy.

The enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis in all cells. The class Ia Escherichia coli RNR consists of two dimeric subunits, α2 and ß2, which form an active but unstable heterodimer of dimers, α2ß2. The structure of the wild-type form of the enzyme has been challenging to study due to the instability of the catalytic complex. A long-range proton-coupled electron-transfer (PCET) pathway facilitates radical migration from the Y122 radical-diiron cofactor in the ß subunit to an active site cysteine, C439, in the α subunit to initiate the RNR chemistry. The PCET reactions and active site chemistry are spectroscopically masked by a rate-limiting, conformational gate. Here, we present a reaction-induced Fourier transform infrared (RIFTIR) spectroscopic method to monitor the mechanism of the active, wild-type RNR α2ß2 complex. This method is employed to obtain new information about conformational changes accompanying RNR catalysis, including the role of carboxylate interactions, deprotonation, and oxidation of active site cysteines, and a detailed description of reversible secondary structural changes. Labeling of tyrosine revealed a conformationally active tyrosine in the ß subunit, assigned to Y356ß, which is part of the intersubunit PCET pathway. New insights into the roles of the inhibitors, azidoUDP and dATP, and the sensitivity of RIFTIR spectroscopy to detect subtle conformational motions arising from protein allostery are also presented.

A Proton Wire Mediates Proton Coupled Electron Transfer from Hydroxyurea and Other Hydroxamic Acids to Tyrosyl Radical in Class Ia Ribonucleotide Reductase.

Proton-coupled electron transfer (PCET) is fundamental to many important biological reactions, including solar energy conversion and DNA synthesis. For example, class Ia ribonucleotide reductases (RNRs) contain a tyrosyl radical-diiron cofactor with one aspartate ligand, D84. The tyrosyl radical, Y122•, in the ß2 subunit acts as a radical initiator and oxidizes an active site cysteine in the α2 subunit. A transient quaternary α2/ß2 complex is induced by substrate and effector binding. The hydroxamic acid, hydroxyurea (HU), reduces Y122• in a PCET reaction involving an electron and proton. This reaction is associated with the loss of activity, a conformational change at Y122, and a change in hydrogen bonding to the Fe1 ligand, D84. Here, we use isotopic labeling, solvent isotope exchange, proton inventories, and reaction-induced Fourier transform infrared (RIFT-IR) spectroscopy to show that the PCET reactions of hydroxamic acids are associated with a characteristic spectrum, which is assignable to electrostatic changes at nonligating aspartate residues. Notably, RIFT-IR spectroscopy reveals this characteristic spectrum when the effects of HU, hydroxylamine, and N-methylhydroxylamine are compared. A large solvent isotope effect is observed for each of the hydroxamic acid reactions, and proton inventories predict that the reactions are associated with the transfer of multiple protons in the transition state. The reduction of Y122• with 4-methoxyphenol does not lead to these characteristic carboxylate shifts and is associated with only a small solvent isotope effect. In addition to studies of the effects of hydroxamic acids on ß2 alone, the reactions involving the quaternary α2ß2 complex were also investigated. HU treatment of the quaternary complex, α2/ß2/ATP/CDP, leads to a similar carboxylate shift spectrum, as observed with ß2 alone. The use of globally labeled 13C chimeras (13C α2, 13C ß2) confirms the assignment. Because the spectrum is sensitive to 13C ß2 labeling, but not 13C α2 labeling, the quaternary complex spectrum is assigned to electrostatic changes in ß2 carboxylate groups. Examination of the ß2 X-ray structure reveals a hydrogen-bonded network leading from the protein surface to Y122. This predicted network includes nonligating aspartates, glutamate ligands to the iron cluster, and predicted crystallographically resolved water molecules. The network is similar when class Ia RNR structures from Escherichia coli, human, and mouse are compared. We propose that the PCET reactions of hydroxamic acids are mediated by a hydrogen-bonded proton wire in the ß2 subunit.

Specific metallo-protein interactions and antimicrobial activity in Histatin-5, an intrinsically disordered salivary peptide.

Histatin-5 (Hst-5) is an antimicrobial, salivary protein that is involved in the host defense system. Hst-5 has been proposed to bind functionally relevant zinc and copper but presents challenges in structural studies due to its disordered conformation in aqueous solution. Here, we used circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy to define metallo-Hst-5 interactions in aqueous solution. A zinc-containing Hst-5 sample exhibits shifted Raman bands, relative to bands observed in the absence of zinc. Based on comparison to model compounds and to a family of designed, zinc-binding beta hairpins, the alterations in the Hst-5 UVRR spectrum are attributed to zinc coordination by imidazole side chains. Zinc addition also shifted a tyrosine aromatic ring UVRR band through an electrostatic interaction. Copper addition did not have these effects. A sequence variant, H18A/H19A, was employed; this mutant has less potent antifungal activity, when compared to Hst-5. Zinc addition had only a small effect on the thermal stability of this mutant. Interestingly, both zinc and copper addition shifted histidine UVRR bands in a manner diagnostic for metal coordination. Results obtained with a K13E/R22G mutant were similar to those obtained with wildtype. These experiments show that H18 and H19 contribute to a zinc binding site. In the H18A/H19A mutant the specificity of the copper/zinc binding sites is lost. The experiments implicate specific zinc binding to be important in the antimicrobial activity of Hst-5.

Tyrosine, cysteine, and proton coupled electron transfer in a ribonucleotide reductase-inspired beta hairpin maquette.

Tyrosine residues act as intermediates in proton coupled electron transfer reactions (PCET) in proteins. For example, in ribonucleotide reductase (RNR), a tyrosyl radical oxidizes an active site cysteine via a 35 Å pathway that contains multiple aromatic groups. When singlet tyrosine is oxidized, the radical becomes a strong acid, and proton transfer reactions, which are coupled with the redox reaction, may be used to control reaction rate. Here, we characterize a tyrosine-containing beta hairpin, Peptide O, which has a cross-strand, noncovalent interaction between its single tyrosine, Y5, and a cysteine (C14). Circular dichroism provides evidence for a thermostable beta-turn. EPR spectroscopy shows that Peptide O forms a neutral tyrosyl radical after UV photolysis at 160 K. Molecular dynamics simulations support a phenolic/SH interaction in the tyrosine singlet and radical states. Differential pulse voltammetry exhibits pH dependence consistent with the formation of a neutral tyrosyl radical and a pKa change in two other residues. A redox-coupled decrease in cysteine pKa from 9 (singlet) to 6.9 (radical) is assigned. At pD 11, picosecond transient absorption spectroscopy after UV photolysis monitors tyrosyl radical recombination via electron transfer (ET). The ET rate in Peptide O is indistinguishable from the ET rates observed in peptides containing a histidine and a cyclohexylalanine (Cha) at position 14. However, at pD 9, the tyrosyl radical decays via PCET, and the decay rate is slowed, when compared to the histidine 14 variant. Notably, the decay rate is accelerated, when compared to the Cha 14 variant. We conclude that redox coupling between tyrosine and cysteine can act as a PCET control mechanism in proteins.

Water Bridges Conduct Sequential Proton Transfer in Photosynthetic Oxygen Evolution.

Proton transfer using water bridges has been observed in bulk water, acid-base reactions, and several proton-translocating biological systems. In the photosynthetic water-oxidizing enzyme, photosystem II (PSII), protons from substrate water are transferred 35 Å from the Mn4CaO5 catalytic site to the chloroplast lumen. This process leads to acidification of the lumen and ATP synthesis. Water oxidation occurs in a flash-induced, five-step S n state cycle; acetate is a chloride-dependent inhibitor of the S2 to S3 step of this cycle. Here, we study the effect of acetate on a previous step of the cycle, the S1 to S2 transition, using reaction-induced infrared spectroscopy. PSII was isolated from spinach, and experiments were conducted at pH 7.5, using 532 nm laser flashes to advance the cycle from the dark-adapted state S1 to the S2 state. Isotope-editing of acetate reveals direct contributions to the S2-minus-S1 infrared spectrum consistent with protonation of bound acetate in PSII. In the acetate-derived S2-minus-S1 PSII spectra, an accompanying decrease in the intensity of a 2830 cm-1 band is observed when compared to the chloride control. The 2830 cm-1 band has been assigned previously to a stretching vibration of an internal, hydrated hydronium ion, W n+. Density functional studies of a catalytic site model predict the spontaneous transfer of a proton from this internal hydronium ion to acetate, when acetate is substituted at a chloride-binding site. Taken together, the results show that the mechanism of PSII proton transfer at pH 7.5 involves proton hopping through an internal, water-containing network.

Structure and Function of Tryptophan-Tyrosine Dyads in Biomimetic ß Hairpins.

Tyrosine-tryptophan (YW) dyads are ubiquitous structural motifs in enzymes and play roles in proton-coupled electron transfer (PCET) and, possibly, protection from oxidative stress. Here, we describe the function of YW dyads in de novo designed 18-mer, ß hairpins. In Peptide M, a YW dyad is formed between W14 and Y5. A UV hypochromic effect and an excitonic Cotton signal are observed, in addition to singlet, excited state (W*) and fluorescence emission spectral shifts. In a second Peptide, Peptide MW, a Y5-W13 dyad is formed diagonally across the strand and distorts the backbone. On a picosecond timescale, the W* excited-state decay kinetics are similar in all peptides but are accelerated relative to amino acids in solution. In Peptide MW, the W* spectrum is consistent with increased conformational flexibility. In Peptide M and MW, the electron paramagnetic resonance spectra obtained after UV photolysis are characteristic of tyrosine and tryptophan radicals at 160 K. Notably, at pH 9, the radical photolysis yield is decreased in Peptide M and MW, compared to that in a tyrosine and tryptophan mixture. This protective effect is not observed at pH 11 and is not observed in peptides containing a tryptophan-histidine dyad or tryptophan alone. The YW dyad protective effect is attributed to an increase in the radical recombination rate. This increase in rate can be facilitated by hydrogen-bonding interactions, which lower the barrier for the PCET reaction at pH 9. These results suggest that the YW dyad structural motif promotes radical quenching under conditions of reactive oxygen stress.

Photoirradiation Generates an Ultrastable 8-Formyl FAD Semiquinone Radical with Unusual Properties in Formate Oxidase.

Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.

Engineering Proton Transfer in Photosynthetic Oxygen Evolution: Chloride, Nitrate, and Trehalose Reorganize a Hydrogen-Bonding Network.

Photosystem II oxidizes water at a Mn4CaO5 cluster. Oxygen evolution is accompanied by proton release through a 35 Šhydrogen-bonding network to the lumen. The mechanism of this proton-transfer reaction is not known, but the reaction is dependent on chloride. Here, vibrational spectroscopy defines the functional properties of the proton-transfer network using chloride, bromide, and nitrate as perturbative agents. As assessed by peptide C═O frequencies, bromide substitution yields a spectral Stark shift because of its increase in ionic radius. Nitrate substitution leads to more complex spectral changes, consistent with an overall increase in hydrogen-bonding interactions with the peptide backbone. The effects are similar to spectral changes previously documented in site-directed mutations in a putative lumenal pathway. Importantly, the effects of nitrate are reversed by the osmolyte, trehalose. Trehalose is known to alter hydrogen-bonding interactions in proteins. Trehalose addition also reverses a shift in an internal hydronium ion signal, consistent with an alteration in its p Ka value and a change in the basicity of bound nitrate. The spectra provide evidence that the proton-transfer pathway contains peptide carbonyl groups, internal water, a hydronium ion, and amino acid side chains. These experiments also show that the proton-transfer pathway functionally adapts to changes in electric field, p Ka, and hydrogen bonding and thereby optimizes proton transfer to the lumen.

Calcium, conformational selection, and redox-active tyrosine YZ in the photosynthetic oxygen-evolving cluster.

In Photosystem II (PSII), YZ (Tyr161D1) participates in radical transfer between the chlorophyll donor and the Mn4CaO5 cluster. Under flashing illumination, the metal cluster cycles among five Sn states, and oxygen is evolved from water. The essential YZ is transiently oxidized and reduced on each flash in a proton-coupled electron transfer (PCET) reaction. Calcium is required for function. Of reconstituted divalent ions, only strontium restores oxygen evolution. YZ is predicted to hydrogen bond to calcium-bound water and to His190D1 in PSII structures. Here, we report a vibrational spectroscopic study of YZ radical and singlet in the presence of the metal cluster. The S2 state is trapped by illumination at 190 K; flash illumination then generates the S2YZ radical. Using reaction-induced FTIR spectroscopy and divalent ion depletion/substitution, we identify calcium-sensitive tyrosyl radical and tyrosine singlet bands in the S2 state. In calcium-containing PSII, two CO stretching bands are detected at 1,503 and 1,478 cm-1 These bands are assigned to two different radical conformers in calcium-containing PSII. At pH 6.0, the 1,503-cm-1 band shifts to 1,507 cm-1 in strontium-containing PSII, and the band is reduced in intensity in calcium-depleted PSII. These effects are consistent with a hydrogen-bonding interaction between the calcium site and one conformer of radical YZ. Analysis of the amide I region indicates that calcium selects for a PCET reaction in a subset of the YZ conformers, which are trapped in the S2 state. These results support the interpretation that YZ undergoes a redox-coupled conformational change, which is calcium dependent.

UV Resonance Raman Study of Apoptosis, Platinum-Based Drugs, and Human Cell Lines.

As a noninvasive molecular analysis technique, ultraviolet resonance Raman (UVRR) spectroscopy represents a label-free method suitable for characterizing biomolecules. Using UVRR spectroscopy, we collected spectral fingerprints of UV absorbing cellular components, including proteins, nucleic acids, and unsaturated lipids. This knowledge was used to guide the assignment of spectra derived from intact human cell lines (i. e., HSC-3 and HaCaT) and from the apoptotic events induced by cisplatin. Notably, a jet-flow system was employed to generate flowing cell suspensions during UVRR measurements, minimizing UV-induced damage. A spectral marker is established based on the ratio of Raman intensities at 1488 and 1655 cm-1 ; this ratio correlates to the level of cell death due to apoptosis. Collectively, this work demonstrates that UVRR spectroscopy is a sensitive and informative probe of cellular physiology and molecular composition. The molecular insight obtained from UVRR measurements can be used to improve understanding of therapeutic treatment and to guide drug development and the choice of therapeutic agents.

Chloride Maintains a Protonated Internal Water Network in the Photosynthetic Oxygen Evolving Complex.

In photosystem II (PSII), water oxidation occurs at a Mn4CaO5 cluster and results in production of molecular oxygen. The Mn4CaO5 cluster cycles among five oxidation states, called Sn states. As a result, protons are released at the metal cluster and transferred through a 35 Å hydrogen-bonding network to the lumen. At 283 K, an infrared band at 2830 cm-1 is assigned to an internal solvated hydronium ion via H218O solvent exchange. This result is similar to a previous report at 263 K. Computations on an oxygen evolving complex model predict that chloride can stabilize a hydronium ion on a network of nine water molecules. In this model, a H3O+ stretching mode at 2738 cm-1 is predicted to shift to higher frequency with bromide and to lower frequency with nitrate substitution. The calculated frequencies were compared to S2-minus-S1 reaction-induced Fourier transform infrared spectra acquired from chloride-, bromide-, or nitrate-containing PSII samples, which were active in oxygen evolution. As predicted, the frequency of the 2830 cm-1 band shifted to higher energy with bromide and to lower energy with nitrate substitution. These results support the conclusion that an internal hydronium ion and chloride play a direct role in an internal proton transfer event during the S1-to-S2 transition.

An Ultraviolet Resonance Raman Spectroscopic Study of Cisplatin and Transplatin Interactions with Genomic DNA.

Ultraviolet resonance Raman (UVRR) spectroscopy is a label-free method to define biomacromolecular interactions with anticancer compounds. Using UVRR, we describe the binding interactions of two Pt(II) compounds, cisplatin (cis-diamminedichloroplatinum(II)) and its isomer, transplatin, with nucleotides and genomic DNA. Cisplatin binds to DNA and other cellular components and triggers apoptosis, whereas transplatin is clinically ineffective. Here, a 244 nm UVRR study shows that purine UVRR bands are altered in frequency and intensity when mononucleotides are treated with cisplatin. This result is consistent with previous suggestions that purine N7 provides the cisplatin-binding site. The addition of cisplatin to DNA also causes changes in the UVRR spectrum, consistent with binding of platinum to purine N7 and disruption of hydrogen-bonding interactions between base pairs. Equally important is that transplatin treatment of DNA generates similar UVRR spectral changes, when compared to cisplatin-treated samples. Kinetic analysis, performed by monitoring decreases of the 1492 cm-1 band, reveals biphasic kinetics and is consistent with a two-step binding mechanism for both platinum compounds. For cisplatin-DNA, the rate constants (6.8 × 10-5 and 6.5 × 10-6 s-1) are assigned to the formation of monofunctional adducts and to bifunctional, intrastrand cross-linking, respectively. In transplatin-DNA, there is a 3.4-fold decrease in the rate constant of the slow phase, compared with the cisplatin samples. This change is attributed to generation of interstrand, rather than intrastrand, adducts. This longer reaction time may result in increased competition in the cellular environment and account, at least in part, for the lower pharmacological efficacy of transplatin.

Tracking Reactive Water and Hydrogen-Bonding Networks in Photosynthetic Oxygen Evolution.

In oxygenic photosynthesis, photosystem II (PSII) converts water to molecular oxygen through four photodriven oxidation events at a Mn4CaO5 cluster. A tyrosine, YZ (Y161 in the D1 polypeptide), transfers oxidizing equivalents from an oxidized, primary chlorophyll donor to the metal center. Calcium or its analogue, strontium, is required for activity. The Mn4CaO5 cluster and YZ are predicted to be hydrogen bonded in a water-containing network, which involves amide carbonyl groups, amino acid side chains, and water. This hydrogen-bonded network includes amino acid residues in intrinsic and extrinsic subunits. One of the extrinsic subunits, PsbO, is intrinsically disordered. This extensive (35 Å) network may be essential in facilitating proton release from substrate water. While it is known that some proteins employ internal water molecules to catalyze reactions, there are relatively few methods that can be used to study the role of water. In this Account, we review spectroscopic evidence from our group supporting the conclusion that the PSII hydrogen-bonding network is dynamic and that water in the network plays a direct role in catalysis. Two approaches, transient electron paramagnetic resonance (EPR) and reaction-induced FT-IR (RIFT-IR) spectroscopies, were used. The EPR experiments focused on the decay kinetics of YZ• via recombination at 190 K and the solvent isotope, pH, and calcium dependence of these kinetics. The RIFT-IR experiments focused on shifts in amide carbonyl frequencies, induced by photo-oxidation of the metal cluster, and on the isotope-based assignment of bands to internal, small protonated water clusters at 190, 263, and 283 K. To conduct these experiments, PSII was prepared in selected steps along the catalytic pathway, the Sn state cycle (n = 0-4). This cycle ultimately generates oxygen. In the EPR studies, S-state dependent changes were observed in the YZ• lifetime and in its solvent isotope effect. The YZ• lifetime depended on the presence of calcium at pH 7.5, but not at pH 6.0, suggesting a two-donor model for PCET. At pH 6.0 or 7.5, barium and ammonia both slowed the rate of YZ• recombination, consistent with disruption of the hydrogen-bonding network. In the RIFT-IR studies of the S state transitions, infrared bands associated with the transient protonation and deprotonation of internal waters were identified by D2O and H218O labeling. The infrared bands of these protonated water clusters, Wn+ (or nH2O(H3O)+, n = 5-6), exhibited flash dependence and were produced during the S1 to S2 and S3 to S0 transitions. Calcium dependence was observed at pH 7.5, but not at pH 6.0. S-state induced shifts were observed in amide C═O frequencies during the S1 to S2 transition and attributed to alterations in hydrogen bonding, based on ammonia sensitivity. In addition, isotope editing of the extrinsic subunit, PsbO, established that amide vibrational bands of this lumenal subunit respond to the S state transitions and that PsbO is a structural template for the reaction center. Taken together, these spectroscopic results support the hypothesis that proton transfer networks, extending from YZ to PsbO, play a functional and dynamic role in photosynthetic oxygen evolution.

Calcium, Ammonia, Redox-Active Tyrosine YZ, and Proton-Coupled Electron Transfer in the Photosynthetic Oxygen-Evolving Complex.

A redox-active tyrosine, YZ (Y161 in the D1 polypeptide), is essential in photosystem II (PSII), which conducts photosynthetic oxygen evolution. On each step of the light-driven oxygen evolving reaction, YZ radical is formed by a chlorophyll cation radical. YZ radical is then reduced by a Mn4CaO5 cluster in a proton coupled electron transfer (PCET) reaction. YZ is hydrogen bonded to His190-D1 and to water molecules in a hydrogen-bonding network, involving calcium. This network is sensitive to disruption with ammonia and to removal and replacement of calcium. Only strontium supports activity. Here, we use electron paramagnetic resonance (EPR) spectroscopy to define the influence of ammonia treatment, calcium removal, and strontium/barium substitution on YZ radical PCET at two pH values. A defined oxidation state of the metal cluster (S2) was trapped by illumination at 190 K. The net reduction and protonation of YZ radical via PCET were monitored by EPR transients collected after a 532 nm laser flash. At 190 K, YZ radical cannot oxidize the Mn4CaO5 cluster and decays on the seconds time scale by recombination with QA-. The overall decay half-time and biexponential fits were used to analyze the results. The reaction rate was independent of pH in control, calcium-reconstituted PSII (Ca-PSII). At pH 7.5, the YZ radical decay rate decreased in calcium-depleted (CD-PSII) and barium/strontium-reconstituted PSII (Ba-PSII, Sr-PSII), relative to Ca-PSII. At pH 6.0, the YZ radical decay rate was not significantly altered in CD-PSII and Sr-PSII but decreased in Ba-PSII. A two-pathway model, involving two competing proton donors with different pKa values, is proposed to explain these results. Ammonia treatment decreased the YZ decay rate in Ca-PSII, Sr-PSII, and CD-PSII, consistent with a reaction that is mediated by the hydrogen-bonding network. However, ammonia treatment did not alter the rate in Ba-PSII. This result is interpreted in terms of the large ionic radius of barium and the elevated pKa of barium-bound water, which are expected to disrupt hydrogen bonding. In addition, evidence for a functional interaction between the S2 protonated water cluster (Wn+) and the YZ proton donation pathway is presented. This interaction is proposed to increase the rate of the YZ PCET reaction.

Redox-Driven Conformational Dynamics in a Photosystem-II-Inspired ß-Hairpin Maquette Determined through Spectroscopy and Simulation.

Tyrosine-based radical transfer plays an important role in photosynthesis, respiration, and DNA synthesis. Radical transfer can occur either by electron transfer (ET) or proton coupled electron transfer (PCET), depending on the pH. Reversible conformational changes in the surrounding protein matrix may control reactivity of radical intermediates. De novo designed Peptide A is a synthetic 18 amino-acid ß-hairpin, which contains a single tyrosine (Y5) and carries out a kinetically significant PCET reaction between Y5 and a cross-strand histidine (H14). In Peptide A, amide II' (CN) changes are observed in the UV resonance Raman (UVRR) spectrum, associated with tyrosine ET and PCET; these bands were attributed previously to a reversible change in secondary structure. Here, we use molecular dynamics simulations to define this conformational change in Peptide A and its H14-to-cyclohexylalanine variant, Peptide C. Three different Y5 charge states, tyrosine (YH), tyrosinate (Y-), and neutral tyrosyl radical (Y·), are considered. The simulations show that Peptide A-YH and A-Y- retain secondary structure and noncovalent interactions, whereas A-Y· is unstable. In contrast, both Peptide C-Y- and Peptide C-Y· are unstable, due to the loss of the Y5-H14 π-π interaction. These simulations are consistent with previous UVRR experimental results on the two ß-hairpins. Furthermore, they demonstrate the ability of simulations using fixed-charge force fields to accurately capture redox-linked conformational dynamics in a ß-strand peptide.

Dynamics of Proton Transfer to Internal Water during the Photosynthetic Oxygen-Evolving Cycle.

In photosynthesis, the light-driven oxidation of water is a sustainable process, which converts solar to chemical energy and produces protons and oxygen. To enable biomimetic strategies, the mechanism of photosynthetic oxygen evolution must be elucidated. Here, we provide information concerning a critical step in the oxygen-evolving, or S-state, cycle. During this S3-to-S0 transition, oxygen is produced, and substrate water binds to the manganese-calcium catalytic site. Our spectroscopic and H218O labeling experiments show that this S3-to-S0 step is associated with the protonation of an internal water cluster in a hydrogen-bonding network, which contains calcium. When compared to the protonated water cluster, formed during a preceding step, the S1-to-S2 transition, the S3-to-S0 hydronium ion is likely to be coordinated by additional water molecules. This evidence shows that internal water and the hydrogen bonding network act as a transient proton acceptor at multiple points in the oxygen-evolving cycle.

Cryogenic Trapping and Isotope Editing Identify a Protonated Water Cluster as an Intermediate in the Photosynthetic Oxygen-Evolving Reaction.

Internal water is known to play a catalytic role in several enzymes. In photosystem II (PSII), water is the substrate. To oxidize water, the PSII Mn4CaO5 cluster or oxygen evolving center (OEC) cycles through five oxidation states, termed Sn states. As reaction products, molecular oxygen is released, and protons are transferred through a ∼25 Šhydrogen-bonded network from the OEC to the thylakoid lumen. Previously, it was reported that a broad infrared band at 2880 cm(-1) is produced during the S1-to-S2 transition and accompanies flash-induced, S state cycling at pH 7.5. Here, we report that when the S2 state is trapped by continuous illumination under cryogenic conditions (190 K), an analogous 2740/2900 cm(-1) band is observed. The frequency depended on the sodium chloride concentration. This band is unambiguously assigned to a normal mode of water by D2(16)O and H2(18)O solvent exchange. Its large, apparent H2(18)O isotope shift, ammonia sensitivity, frequency, and intensity support assignment to a stretching vibration of a hydronium cation, H3O(+), in a small, protonated internal water cluster, nH2O(H3O(+)). Water OH stretching bands, which may be derived from the hydration shell of the hydronium ion, are also identified. Using the 2740 cm(-1) infrared marker, the results of calcium depletion and strontium reconstitution on the protonated water cluster are found to be pH dependent. This change is attributed to protonation of an amino acid side chain and a possible change in nH2O(H3O)(+) localization in the hydrogen-bonding network. These results are consistent with an internal water cluster functioning as a proton acceptor and an intermediate during the S1-to-S2 transition. Our experiments demonstrate the utility of this infrared signal as a novel functional probe in PSII.

Proton-Coupled Electron Transfer and a Tyrosine-Histidine Pair in a Photosystem II-Inspired ß-Hairpin Maquette: Kinetics on the Picosecond Time Scale.

Photosystem II (PSII) and ribonucleotide reductase employ oxidation and reduction of the tyrosine aromatic ring in radical transport pathways. Tyrosine-based reactions involve either proton-coupled electron transfer (PCET) or electron transfer (ET) alone, depending on the pH and the pKa of tyrosine's phenolic oxygen. In PSII, a subset of the PCET reactions are mediated by a tyrosine-histidine redox-driven proton relay, YD-His189. Peptide A is a PSII-inspired ß-hairpin, which contains a single tyrosine (Y5) and histidine (H14). Previous electrochemical characterization indicated that Peptide A conducts a net PCET reaction between Y5 and H14, which have a cross-strand π-π interaction. The kinetic impact of H14 has not yet been explored. Here, we address this question through time-resolved absorption spectroscopy and 280-nm photolysis, which generates a neutral tyrosyl radical. The formation and decay of the neutral tyrosyl radical at 410 nm were monitored in Peptide A and its variant, Peptide C, in which H14 is replaced by cyclohexylalanine (Cha14). Significantly, both electron transfer (ET, pL 11, L = lyonium) and PCET (pL 9) were accelerated in Peptide A and C, compared to model tyrosinate or tyrosine at the same pL. Increased electronic coupling, mediated by the peptide backbone, can account for this rate acceleration. Deuterium exchange gave no significant solvent isotope effect in the peptides. At pL 9, but not at pL 11, the reaction rate decreased when H14 was mutated to Cha14. This decrease in rate is attributed to an increase in reorganization energy in the Cha14 mutant. The Y5-H14 mechanism in Peptide A is reminiscent of proton- and electron-transfer events involving YD-H189 in PSII. These results document a mechanism by which proton donors and acceptors can regulate the rate of PCET reactions.

A tyrosine-tryptophan dyad and radical-based charge transfer in a ribonucleotide reductase-inspired maquette.

In class 1a ribonucleotide reductase (RNR), a substrate-based radical is generated in the α2 subunit by long-distance electron transfer involving an essential tyrosyl radical (Y122O·) in the ß2 subunit. The conserved W48 ß2 is ∼10 Å from Y122OH; mutations at W48 inactivate RNR. Here, we design a beta hairpin peptide, which contains such an interacting tyrosine-tryptophan dyad. The NMR structure of the peptide establishes that there is no direct hydrogen bond between the phenol and the indole rings. However, electronic coupling between the tyrosine and tryptophan occurs in the peptide. In addition, downshifted ultraviolet resonance Raman (UVRR) frequencies are observed for the radical state, reproducing spectral downshifts observed for ß2. The frequency downshifts of the ring and CO bands are consistent with charge transfer from YO· to W or another residue. Such a charge transfer mechanism implies a role for the ß2 Y-W dyad in electron transfer.