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 exhibits a significant solvent isotope effect, and the rate of recombination and the solvent isotope effect are 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.

Proton coupled electron transfer (PCET) reactions are important in many biological processes. Tyrosine oxidation/reduction can play a critical role in facilitating these reactions. Two examples are photosystem II (PSII) and ribonucleotide reductase (RNR). RNR is essential in DNA synthesis in all organisms. In E. coli RNR, a tyrosyl radical, Y122•, is required as a radical initiator. Photosystem II (PSII) generates molecular oxygen from water. In PSII, an essential tyrosyl radical, YZ•, oxidizes the oxygen evolving center. However, the mechanisms, by which the extraordinary oxidizing power of the tyrosyl radical is controlled, are not well understood. This is due to the difficulty in acquiring high-resolution structural information about the radical state. Spectroscopic approaches, such as EPR and UV resonance Raman (UVRR), can give new information. Here, we discuss EPR studies of PCET and the PSII YZ radical. We also present UVRR results, which support the conclusion that Y122 undergoes an alteration in ring and backbone dihedral angle when it is oxidized. This conformational change results in a loss of hydrogen bonding to the phenolic oxygen. Our analysis suggests that access of water is an important factor in determining tyrosyl radical lifetime and function. TOC graphic

In photosynthetic oxygen evolution, redox active tyrosine Z (YZ) plays an essential role in proton-coupled electron transfer (PCET) reactions. Four sequential photooxidation reactions are necessary to produce oxygen at a Mn(4)CaO(5) cluster. The sequentially oxidized states of this oxygen-evolving cluster (OEC) are called the S(n) states, where n refers to the number of oxidizing equivalents stored. The neutral radical, YZ•, is generated and then acts as an electron transfer intermediate during each S state transition. In the X-ray structure, YZ, Tyr161 of the D1 subunit, is involved in an extensive hydrogen bonding network, which includes calcium-bound water. In electron paramagnetic resonance experiments, we measured the YZ• recombination rate, in the presence of an intact Mn(4)CaO(5) cluster. We compared the S(0) and S(2) states, which differ in Mn oxidation state, and found a significant difference in the YZ• decay rate (t(1/2) = 3.3 ± 0.3 s in S(0); t(1/2) = 2.1 ± 0.3 s in S(2)) and in the solvent isotope effect (SIE) on the reaction (1.3 ± 0.3 in S(0); 2.1 ± 0.3 in S(2)). Although the YZ site is known to be solvent accessible, the recombination rate and SIE were pH independent in both S states. To define the origin of these effects, we measured the YZ• recombination rate in the presence of ammonia, which inhibits oxygen evolution and disrupts the hydrogen bond network. We report that ammonia dramatically slowed the YZ• recombination rate in the S(2) state but had a smaller effect in the S(0) state. In contrast, ammonia had no significant effect on YD•, the stable tyrosyl radical. Therefore, the alterations in YZ• decay, observed with S state advancement, are attributed to alterations in OEC hydrogen bonding and consequent differences in the YZ midpoint potential/pK(a). These changes may be caused by activation of metal-bound water molecules, which hydrogen bond to YZ. These observations document the importance of redox control in proton-coupled electron transfer reactions.

Photosystem I (PSI) is one of the two membrane-associated reaction centers involved in oxygenic photosynthesis. In photosynthesis, solar energy is converted to chemical energy in the form of a transmembrane charge separation. PSI oxidizes cytochrome c 6 or plastocyanin and reduces ferredoxin. In cyanobacterial PSI, there are 10 tryptophan residues with indole side chains located less than 10 Å from the electron transfer cofactors. In this study, we apply pump-probe difference UV resonance Raman (UVRR) spectroscopy to acquire the spectrum of aromatic amino acids in cyanobacterial PSI. This UVRR technique allows the use of the tryptophan vibrational spectrum as a reporter for structural changes, which are linked to PSI electron transfer reactions. Our results show that photo-oxidation of the chlorophyll a/a′ heterodimer, P 700 , causes shifts in the vibrational frequencies of two or more tryptophan residues. Similar perturbations of tryptophan are observed when P 700 is chemically oxidized. The observed spectral frequencies suggest that the perturbed tryptophan side chains are only weakly or not hydrogen bonded and are located in an environment in which there is steric repulsion. The direction of the spectral shifts is consistent with an oxidationinduced increase in dielectric constant or a change in hydrogen bonding. To explain our results, the perturbation of tryptophan residues must be linked to a PSI conformational change, which is, in turn, driven by P 700 oxidation.Long distance electron transfer occurs in many important biological processes, including enzymes involved in mitochondrial electron transfer, photosynthetic energy transduction, and DNA synthesis. 1 In these systems, the protein environment provides a responsive matrix, which controls and regulates the reactions.2 For example, the phenol and indole side chains of tyrosine3 and tryptophan4, respectively, are redox-active in some enzymes. In addition, the tryptophan side chain can provide an electron tunneling bridge. 4 Previously, changes in the tryptophan ultraviolet (UV) absorption spectrum have been used as a probe of protein dynamics. 5, 6 Work on bacteriorhodopsin and the bacterial reaction center suggests that an initial light absorption event can alter protein conformation. Such pre-tuning of the protein matrix can play an important role in the control of subsequent enzymatic reactions. 5 , 6 PSI is one of the two membrane-associated reaction centers involved in oxygenic photosynthesis. 7 In photosynthesis, solar energy is converted to chemical energy in the form of a transmembrane charge separation. PSI catalyzes the transfer of an electron from plastocyanin or a cytochrome c 6 to ferredoxin. 8 Electron transfer is initiated by photoexcitation of the primary electron donor, which is most likely a monomeric accessory chlorophyll (chl). Figure 1, the electron is then transferred through a series of acceptor molecules, which are named A 0 (chlorophyll a), A 1 (phylloquinone), F x (iron sulfur cluster), F A (iron sulfur cluster), an...

Photosystem I (PSI) is a multisubunit protein complex which carries out light-induced, transmembrane charge separation in oxygenic photosynthesis. In PSI, the electron-transfer pathway consists of chlorophyll and phylloquinone molecules, as well as iron-sulfur clusters. There are two phylloquinone molecules, which are located in structurally symmetric positions in the reaction center. It has been proposed that both phylloquinone molecules are active as the A1 secondary electron acceptor in bidirectional electron-transfer reactions. The PSI A1 acceptors are of interest because they have the lowest reduction potential of any quinone found in nature. In this work using light-induced FT-IR spectroscopy, isotope-edited spectra are presented, which attribute vibrational bands to the carbonyl stretching vibrations of A1 and A1- and the quinoid ring stretching vibration of A1. Bands are assigned by comparison with hybrid Hartee-Fock density functional calculations, which predict vibrational frequencies, amplitudes, and isotope shifts for the phylloquinone singlet and radical anion states. The results are consistent with an environmental interaction increasing the frequency of the singlet CO vibration and decreasing the frequency of the anion radical CO vibration, relative to model compounds. This environmental interaction may be the asymmetric hydrogen bond to A1/A1-, electrostatic interactions with charged amino acid side chains, or a pi-pi interaction with the indole ring of a nearby tryptophan. Such differential effects on the structure of A1 and A1- may be associated with a destabilization of the anion radical. These studies give novel information concerning the effect of the protein matrix on the PSI electron-transfer cofactor.