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In biological water oxidation, a redox-active tyrosine residue (D1-Tyr161 or Y Z ) mediates electron transfer between the Mn 4 CaO 5 cluster of the oxygen-evolving complex and the charge-separation site of photosystem II (PSII), driving the cluster through progressively higher oxidation states S i ( i = 0-4). In contrast to lower S-states (S 0 , S 1 ), in higher S-states (S 2 , S 3 ) of the Mn 4 CaO 5 cluster, Y Z cannot be oxidized at cryogenic temperatures due to the accumulation of positive charge in the S 1 → S 2 transition. However, oxidation of Y Z by illumination of S 2 at 77-190 K followed by rapid freezing and charge recombination between Y Z • and the plastoquinone radical Q A •- allows trapping of an S 2 variant, the so-called S 2 trapped state (S 2 t ), that is capable of forming Y Z • at cryogenic temperature. To identify the differences between the S 2 and S 2 t states, we used the S 2 t Y Z • intermediate as a probe for the S 2 t state and followed the S 2 t Y Z • /Q A •- recombination kinetics at 10 K using time-resolved electron paramagnetic resonance spectroscopy in H 2 O and D 2 O. The results show that while S 2 t Y Z • /Q A •- recombination can be described as pure electron transfer occurring in the Marcus inverted region, the S 2 t → S 2 reversion depends on proton rearrangement and exhibits a strong kinetic isotope effect. This suggests that Y Z oxidation in the S 2 t state is facilitated by favorable proton redistribution in the vicinity of Y Z , most likely within the hydrogen-bonded Y Z -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.

An artificial oxygen-evolving complex (OEC) contains a tetranuclear Co III 4 O 4 cubic cluster ligated with acetate and pyridine molecules, a light-activated Ru(bpy) 3 2+ moiety (bpy = 2,2'-bipyridine), and the sacrificial electron acceptor S 2 configuration of the Co(II) species. The measurement of the EPR spectrum at a wider magnetic field range in comparison to that reported recently shows the presence of an additional signal that contributes to the spectrum of the Co(II) center. The theoretical simulation of this spectrum reveals that an isotropic g value and considerably small zero-field splitting parameters describe the high-spin Co(II) ion in a unique way, which supports a tetrahedral crystal field symmetry. On the basis of the spin-Hamiltonian parameters, the looping transitions that lead to the experimental EPR signals are determined. Additionally, a possible role of the symmetry of the Co(II) species and a proposed model that explains its formation during the O 8 2- side. A recent EPR investigation of this system showed the formation of the high-spin Co(II) ion under visible-illumination conditions. It has been supported that this center originates from the cluster and is involved in the oxygen-evolving process. The present report is focused on the further characterization of the high-spin d 7 configuration of the Co(II) species. The measurement of the EPR spectrum at a wider magnetic field range in comparison to that reported recently shows the presence of an additional signal that contributes to the spectrum of the Co(II) center. The theoretical simulation of this spectrum reveals that an isotropic g value and considerably small zero-field splitting parameters describe the high-spin Co(II) ion in a unique way, which supports a tetrahedral crystal field symmetry. On the basis of the spin-Hamiltonian parameters, the looping transitions that lead to the experimental EPR signals are determined. Additionally, a possible role of the symmetry of the Co(II) species and a proposed model that explains its formation during the O 2 -evolving process of the Co 4 O 4 cubic molecules are discussed.

IspG is a [4Fe-4S] cluster-containing protein, and the [4Fe-4S](+) species is proposed to be the catalytically relevant species. However, attempts reported in the literature failed to detect the [4Fe-4S](+) species. In this study, using a potent reduction system, we have successfully detected the [4Fe-4S](+) species with X-band EPR spectroscopy. In addition, we have improved the Escherichia coli IspG activity to 550 nmol min(-1) mg(-1), which is approximately 20-fold greater than that of the NADPH-Fpr-FldA system in the literature.

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.

Tyr Z of photosystem II mediates electron transfer from the water splitting site, a Mn4Ca cluster, to the specialized chlorophyll assembly P680. Due to its proton-limited redox properties and the proximity to the Mn cluster, it is thought to play a critical role in the proton-coupled electron transfer reactions that constitute the four-step oxidation mechanism (so-called S-state transitions) of water to molecular oxygen. Spectroscopic evidence for the Tyr Z radical has been scarce in intact preparations (it is difficult to probe it optically, and too short-lived for EPR characterization) until recently. Advances in recent years have allowed the trapping at liquid helium temperatures and EPR characterization of metalloradical intermediates, attributed to tyrosyl Z* magnetically interacting with the Mn cluster. We have extended these studies and examined the evolution of the spectra of five intermediates: S0YZ*, S0YZ* (with 5% MeOH), S1YZ*, S2YZ*, and S2YZ* (with 5% MeOH) in the temperature range of 11-230 K. A rapid-scan EPR method has been applied at elevated temperatures. The tyrosyl radical decouples progressively from Mn, as the Mn relaxation rate increases with an increase in temperature. Above approximately 100 K, the spectra collapse to the unperturbed spectrum of Tyr Z*, which is found to be somewhat broader than that of the stable Tyr D* radical. This study provides a simple means for recording the spectrum of Tyr Z* and extends earlier observations that link the photochemistry at liquid helium temperatures to the photochemistry at temperatures that support S-state transitions.

Photosystem II preparations poised in the S(2)...Q(A) state produce no detectable intermediate during straightforward illumination at liquid helium temperatures. However, upon flash illumination in the range of 77-190 K, they produce a transient state which at -10 degrees C advances to S(3) or after rapid cooling to 10 K gives rise to a 116 G wide metalloradical EPR signal. The latter decays with half-times on the order of a few minutes, presumably by charge recombination, and can be regenerated repeatedly by illumination at 10 K. The constraints for Tyr Z oxidation are attributed to the presence of excess positive charge in S(2). Elevated temperatures are required presumably to overcome a thermal barrier in the deprotonation of Tyr Z(+) or most likely to allow secondary proton transfer away from the base partner of Tyr Z. Treatment with 5% (v/v) MeOH appears to remove the constraints for Tyr Z oxidation, and a 160 G wide metalloradical EPR signal is produced by illumination at 10 K, which decays with a half-time of ca. 80 s. Formation of the metalloradical signals is accompanied by reversible changes in the Mn multiline signal. The intermediates are assigned to Tyr Z(*) magnetically interacting with the Mn cluster in S(2), S(2)Y(Z)(*). A molecular model which extends an earlier suggestion and provides a plausible explanation of a number of observations, including the binding of small molecules to the Mn cluster, is presented.