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The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from Tyr(Z) to P(680)(*+) has been decreased by approximately 80 meV by mutating the axial ligand of P(680), and that for proton transfer upon oxidation of Tyr(Z) by substituting a 3-fluorotyrosine (3F-Tyr(Z)) for Tyr(Z). In Mn-depleted Photosystem II, the dependence upon pH of the oxidation rates of Tyr(Z) and 3F-Tyr(Z) were found to be similar. However, in the pH range where the phenolic hydroxyl of Tyr(Z) is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-Tyr(Z) is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al. J. Am. Chem. Soc. 2006, 128,1569-1579). Thus, when the phenol of Y(Z) acts as a H-bond donor, its oxidation by P(680)(*+) is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of Tyr(Z) has been proposed to occur concertedly with the electron transfer to P(680)(*+). This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.

Superoxide dismutases (SODs) are proteins specialized in the depletion of superoxide from the cell through disproportionation of this anion into oxygen and hydrogen peroxide. We have used high-field electron paramagnetic resonance (HFEPR) to test a two-site binding model for the interaction of manganese-SODs with small anions. Because tyrosine-34 was thought to act as a gate between these two sites in this model, a tyrosine to phenylalanine mutant of the superoxide dismutase from R. capsulatus was constructed. Although the replacement slightly reduced activity, HFEPR measurements demonstrated that the electronic structure of the Mn(II) center was unaffected by the mutation. In contrast, the mutation had a profound effect on the interactions of fluoride and azide with the Mn(II) center. It was concluded that the absence of tyrosine-34 prevented the close approach of these anions to the metal ion. This mutation also enhanced the formation of a hexacoordinated water-Mn(II)SOD complex at low temperatures. Together, these results showed that the role of Y34 is unlikely to involve redox tuning but rather is important in regulating the equilibria between the anionic substrate in solution and the two binding sites near the metal. These observations further supported the originally proposed mutually exclusive two-binding-site model.

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.

The coupling of proton chemistry with redox reactions is important in many enzymes and is central to energy transduction in biology. However, the mechanistic details are poorly understood. Here, we have studied tyrosine oxidation, a reaction in which the removal of one electron from the amino acid is linked to the release of its phenolic proton. Using the unique photochemical properties of photosystem II, it was possible to oxidize the tyrosine at 1.8 K, a temperature at which proton and protein motions are limited. The state formed was detected by high magnetic field EPR as a high-energy radical intermediate trapped in an unprecedentedly electropositive environment. Warming of the protein allows this state to convert to a relaxed, stable form of the radical. The relaxation event occurs at 77 K and seems to involve proton migration and only a very limited movement of the protein. These reactions represent a stabilization process that prevents the back-reaction and determines the reactivity of the radical.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.

The local electrostatic environment plays a critical role in determining the physicochemical properties of reactive radicals in proteins. High-field electron paramagnetic resonance (HF-EPR) spectroscopy has been used to determine the sensitivity of the tyrosyl radical g-values to local electrostatic environment. Site-specific mutants of ribonucleotide reductase from Escherichia coli were used to study the effect of introducing a charge group on the HF-EPR spectrum of the stable tyrosyl (Y122) radical. The changes affected by the mutations were small, but measurable. Mutation of isoleucine-74 to an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine interactions. Similar effects were observed for the mutation of valine-136 to an arginine (V136R) or asparagine (V136N). For five or six mutants studied, the g(x)() component of the g-tensor was distributed. For the isoleucine-74 to lysine (I74K) and leucine-77 to phenylalanine (L77F) mutants, a shift of 1 x 10(-)(4) in g(x)() value was also detected. For the I74K mutant, it is shown that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of the nearby diiron center. For the L77F mutant, the shift is induced by the diiron center. Using existing tyrosyl radical g-tensor measurements, we have developed a simple effective charge model that allows us to rationalize the effect of the local electrostatic environments in a number of proteins.

The radical forms of two cofactors and an amino acid in the photosystem II (PS II) reaction center were studied by using high-field EPR both in frozen solution and in oriented multilayers. Their orientation with respect to the membrane was determined by using one-dimensionally oriented samples. The ring plane of the stable tyrosyl radical, Y(D)(*), makes an angle of 64 degrees +/- 5 degrees with the membrane plane, and the C-O direction is tilted by 72 degrees +/- 5 degrees in the plane of the radical compared to the membrane plane. The semiquinone, Q(A)(*)(-), generated by chemical reduction in samples lacking the non-heme iron, has its ring plane at an angle of 72 degrees +/- 5 degrees to the membrane plane, and the O-O axis is tilted by 21 degrees +/- 5 degrees in the plane of the quinone compared to the membrane plane. This orientation is similar to that of Q(A)(*)(-) in purple bacteria reaction centers except for the tilt angle which is slightly bigger. The pheophytin anion was generated by photoaccumulation under reducing conditions. Its ring plane is almost perpendicular to the membrane with an angle of 70 degrees +/- 5 degrees with respect to the membrane plane. This is very similar to the orientation of the pheophytin in purple bacteria reaction centers. The position of the g tensor with respect to the molecule is tentatively assigned for the anion radical on the basis of this comparison. In this work, the treatment of orientation data from EPR spectroscopy applied to one-dimensionally oriented multilayers is examined in detail, and improvements over previous approaches are given.

Ribonucleotide reductase (RNR) is a key enzyme for DNA synthesis since it provides cells with deoxyribonucleotides, the DNA precursors. Class I alpha2beta2 RNRs contain a dinuclear iron center and an essential tyrosyl radical in the beta2 component (protein R2). This is also true for the purified protein R2 of Mycobacterium tuberculosis RNR, as shown by iron analysis, light absorption and EPR spectroscopy. EPR spectroscopy at 286 GHz revealed a high g(x) value, suggesting that the radical is not hydrogen bonded, as in other prokaryotic R2s and in contrast with eukaryotic R2s (from Arabidopsis thaliana and mouse). Furthermore, it proved to be very resistant to scavenging by a variety of phenols and thiols and by hydroxyurea, similar to the Escherichia coli radical. By comparison, the plant and mouse radicals are very sensitive to drugs such as resveratrol and 2-thiophenthiol. The radical from M. tuberculosis RNR does not seem to be an appropriate target for new antituberculous agents.

A 245 GHz 8.7 T high-field EPR study of tyrosine-D (TyrD zero) and tyrosine-Z (TyrZ zero) radicals of photosystem II (PSII) from Synechocystis PCC 6803 was carried out. Identical principal g values for the wild-type Synechocystis and spinach TyrD zero showed that the two radicals were in similar electrostatic environments. By contrast, the principal g values of the TyrD zero in the D2-His189Gln mutant of Synechocystis were different from those of the wild-type and spinach radicals and were similar to those of the tyrosyl radical in ribonucleotide reductase. These comparisons indicate that the D2-His189Gln mutant TyrD zero is not hydrogen-bonded or is only weakly so. The HF-EPR spectrum of TyrZ zero was obtained from the D2-Tyr160Phe mutant that lacks TyrD zero. The principal g values were nearly identical to those of the wild-type TyrD zero. The low-field edge of the TyrZ zero spectrum was much broader than at the other two principal g values and was also much broader than the TyrD zero spectrum. From the identical g values and previous work on tyrosyl radical g values [Un S., Atta M., Fontecave, M., & Rutherford, A. W. (1995) J. Am. Chem. Soc. 117, 10713-10719], it was concluded that TyrZ zero, like TyrD zero, is hydrogen-bonded The broadness of the gx component was interpreted as a distribution in strength of the hydrogen-bonding due to disorder in the protein environment about TyrZ zero.

The 4 K 245-GHz/8.7-T electron paramagnetic resonance spectrum of the stable tyrosyl radical in photosystem II, known as TyrD., has been measured. Illumination at 200 K enhances the signal intensity of TyrD. by a factor of > 40 compared to the signal obtained from dark-adapted samples. This signal enhancement and the unusual line shape of the TyrD. resonance result from the magnetic dipolar coupling of the radical to the manganese cluster involved in oxygen evolution. The relative angular orientation of the manganese cluster with respect to TyrD. has been determined from line-shape analysis. The resonance arising from TyrD. in Tris-washed manganese-free photosystem II sample is also distorted. This effect probably originates from the influence of the nonheme iron on the spin relaxation of the tyrosyl radical. The relative angular orientation of the nonheme iron has also been determined. Oriented samples were used to determine the angular orientation of TyrD. with respect to the membrane plane. Combining angular data with published distances, we have constructed a three-dimensional picture of the relative positions of TyrD., the manganese cluster, and the nonheme iron. The data suggest a more symmetrical placement of the manganese relative to TyrD. and TyrZ, the tyrosine involved in electron transfer, than is usually assumed in current models of photosystem II.