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Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1Å) and Mn,Fe(III)Fe(II) species (~3.3-3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(•), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(•)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.

Metalloradical EPR signals have been found in intact Photosystem II at cryogenic temperatures. They reflect the light-driven formation of the tyrosine Z radical (Y(Z)) in magnetic interaction with the CaMn(4) cluster in a particular S state. These so-called split EPR signals, induced at cryogenic temperatures, provide means to study the otherwise transient Y(Z) and to probe the S states with EPR spectroscopy. In the S(0) and S(1) states, the respective split signals are induced by illumination of the sample in the visible light range only. In the S(3) state the split EPR signal is induced irrespective of illumination wavelength within the entire 415-900nm range (visible and near-IR region) [Su, J. H., Havelius, K. G. V., Ho, F. M., Han, G., Mamedov, F., and Styring, S. (2007) Biochemistry 46, 10703-10712]. An important question is whether a single mechanism can explain the induction of the Split S(3) signal across the entire wavelength range or whether wavelength-dependent mechanisms are required. In this paper we confirm that the Y(Z) radical formation in the S(1) state, reflected in the Split S(1) signal, is driven by P680-centered charge separation. The situation in the S(3) state is different. In Photosystem II centers with pre-reduced quinone A (Q(A)), where the P680-centered charge separation is blocked, the Split S(3) EPR signal could still be induced in the majority of the Photosystem II centers using both visible and NIR (830nm) light. This shows that P680-centered charge separation is not involved. The amount of oxidized electron donors and reduced electron acceptors (Q(A)(-)) was well correlated after visible light illumination at cryogenic temperatures in the S(1) state. This was not the case in the S(3) state, where the Split S(3) EPR signal was formed in the majority of the centers in a pathway other than P680-centered charge separation. Instead, we propose that one mechanism exists over the entire wavelength interval to drive the formation of the Split S(3) signal. The origin for this, probably involving excitation of one of the Mn ions in the CaMn(4) cluster in Photosystem II, is discussed.

The redox-active tyrosine residue (Y-Z) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals reflecting the light-induced Y-Z c ...

Water oxidation in photosystem II is catalyzed by the CaMn(4) cluster. The electrons extracted from the CaMn(4) cluster are transferred to P(680)(+) via the redox-active tyrosine residue D1-Tyr161 (Y(Z)). The oxidation of Y(Z) is coupled to a deprotonation creating the neutral radical Y(Z)(*). Light-induced oxidation of Y(Z) is possible down to extreme temperatures. This can be observed as a split EPR signal from Y(Z)(*) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active PSII. Here we have used the split S(0) EPR signal to study the mechanism of Y(Z) oxidation at 5 K in the S(0) state. The state of the hydrogen bond between Y(Z) and its proposed hydrogen bond partner D1-His190 is investigated by varying the pH. The split S(0) EPR signal was induced by illumination at 5 K between pH 3.9 and pH 9.0. Maximum signal intensity was observed between pH 6 and pH 7. On both the acidic and alkaline sides the signal intensity decreased with the apparent pK(a)s (pK(app)) approximately 4.8 and approximately 7.9, respectively. The illumination protocol used to induce the split S(0) EPR signal also induces a mixed radical signal in the g approximately 2 region. One part of this signal decays with similar kinetics as the split S(0) EPR signal ( approximately 3 min, at 5 K) and is easily distinguished from a stable radical originating from Car/Chl. We suggest that this fast-decaying radical originates from Y(Z)(*). The pH dependence of the light-induced fast-decaying radical was measured in the same pH range as for the split S(0) EPR signal. The pK(app) for the light-induced fast-decaying radical was identical at acidic pH ( approximately 4.8). At alkaline pH the behavior was more complex. Between pH 6.6 and pH 7.7 the signal decreased with pK(app) approximately 7.2. However, above pH 7.7 the induction of the radical species was pH independent. We compare our results with the pH dependence of the split S(1) EPR signal induced at 5 K and the S(0) --> S(1) and S(1) --> S(2) transitions at room temperature. The result allows mechanistic conclusions concerning differences between the hydrogen bond pattern around Y(Z) in the S(0) and S(1) states.

The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.

High-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection, supported by density functional theory calculations (XAES-DFT), was used to study a model complex, ([Fe(2)(μ-adt)(CO)(4)(PMe(3))(2)] (1, adt = S-CH(2)-(NCH(2)Ph)-CH(2)-S), of the [FeFe] hydrogenase active site. For 1 in powder material (1(powder)), in MeCN solution (1'), and in its three protonated states (1H, 1Hy, 1HHy; H denotes protonation at the adt-N and Hy protonation of the Fe-Fe bond to form a bridging metal hydride), relations between the molecular structures and the electronic configurations were determined. EXAFS analysis and DFT geometry optimization suggested prevailing rotational isomers in MeCN, which were similar to the crystal structure or exhibited rotation of the (CO) ligands at Fe1 (1(CO), 1Hy(CO)) and in addition of the phenyl ring (1H(CO,Ph), 1HHy(CO,Ph)), leading to an elongated solvent-exposed Fe-Fe bond. Isomer formation, adt-N protonation, and hydride binding caused spectral changes of core-to-valence (pre-edge of the Fe K-shell absorption) and of valence-to-core (Kß(2,5) emission) electronic transitions, and of Kα RIXS data, which were quantitatively reproduced by DFT. The study reveals (1) the composition of molecular orbitals, for example, with dominant Fe-d character, showing variations in symmetry and apparent oxidation state at the two Fe ions and a drop in MO energies by ~1 eV upon each protonation step, (2) the HOMO-LUMO energy gaps, of ~2.3 eV for 1(powder) and ~2.0 eV for 1', and (3) the splitting between iron d(z(2)) and d(x(2)-y(2)) levels of ~0.5 eV for the nonhydride and ~0.9 eV for the hydride states. Good correlations of reduction potentials to LUMO energies and oxidation potentials to HOMO energies were obtained. Two routes of facilitated bridging hydride binding thereby are suggested, involving ligand rotation at Fe1 for 1Hy(CO) or adt-N protonation for 1HHy(CO,Ph). XAES-DFT thus enables verification of the effects of ligand substitutions in solution for guided improvement of [FeFe] catalysts.

The active site for hydrogen production in [FeFe] hydrogenase comprises a diiron unit. Bioinorganic chemistry has modeled important features of this center, aiming at mechanistic understanding and the development of novel catalysts. However, new assays are required for analyzing the effects of ligand variations at the metal ions. By high-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection (XAS/XES = XAES) and density functional theory (DFT), we studied an asymmetrically coordinated [FeFe] model complex, [(CO)(3)Fe(I)1-(bdtCl(2))-Fe(I)2(CO)(Ph(2)P-CH(2)-NCH(3)-CH(2)-PPh(2))] (1, bdt = benzene-1,2-dithiolate), in comparison to iron-carbonyl references. Kβ emission spectra (Kβ(1,3), Kβ') revealed the absence of unpaired spins and the low-spin character for both Fe ions in 1. In a series of low-spin iron compounds, the Kβ(1,3) energy did not reflect the formal iron oxidation state, but it decreases with increasing ligand field strength due to shorter iron-ligand bonds, following the spectrochemical series. The intensity of the valence-to-core transitions (Kβ(2,5)) decreases for increasing Fe-ligand bond length, certain emission peaks allow counting of Fe-CO bonds, and even molecular orbitals (MOs) located on the metal-bridging bdt group of 1 contribute to the spectra. As deduced from 3d → 1s emission and 1s → 3d absorption spectra and supported by DFT, the HOMO-LUMO gap of 1 is about 2.8 eV. Kβ-detected XANES spectra in agreement with DFT revealed considerable electronic asymmetry in 1; the energies and occupancies of Fe-d dominated MOs resemble a square-pyramidal Fe(0) for Fe1 and an octahedral Fe(II) for Fe2. EXAFS spectra for various Kβ emission energies showed considerable site-selectivity; approximate structural parameters similar to the crystal structure could be determined for the two individual iron atoms of 1 in powder samples. These results suggest that metal site- and spin-selective XAES on [FeFe] hydrogenase protein and active site models may provide a powerful tool to study intermediates under reaction conditions.

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-A Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.

Ten square-based pyramidal molybdenum complexes with different sulfur donor ligands, that is, a variety of dithiolenes and sulfides, were prepared, which mimic coordination motifs of the molybdenum cofactors of molybdenum-dependent oxidoreductases. The model compounds were investigated by Mo K-edge X-ray absorption spectroscopy (XAS) and (with one exception) their molecular structures were analyzed by X-ray diffraction to derive detailed information on bond lengths and geometries of the first coordination shell of molybdenum. Only small variations in Mo=O and Mo-S bond lengths and their respective coordination angles were observed for all complexes including those containing Mo(CO) 2 or Mo(μ-S)2Mo motifs. XAS analysis (edge energy) revealed higher relative oxidation levels in the molybdenum ion in compounds with innocent sulfur-based ligands relative to those in dithiolene complexes, which are known to exhibit noninnocence, that is, donation of substantial electron density from ligand to metal. In addition, longer average Mo-S and Mo=O bonds and consequently lower ν(Mo=O) stretching frequencies in the IR spectra were observed for complexes with dithiolene-derived ligands. The results emphasize that the noninnocent character of the dithiolene ligand influences the electronic structure of the model compounds, but does not significantly affect their metal coordination geometry, which is largely determined by the Mo(IV) or (V) ion itself. The latter conclusion also holds for the molybdenum site geometries in the oxidized MoVI cofactor of DMSO reductase and the reduced MoIV cofactor of arsenite oxidase. The innocent behavior of the dithiolene molybdopterin ligands observed in the enzymes is likely to be related to cofactor-protein interactions. (Less)

YedY from Escherichia coil is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of similar to 2.4 angstrom), and one (axial) oxo ligand (Mo=O, similar to 1.7 angstrom). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (similar to 2.1 angstrom) or possibly to a shorter M-O(-) bond. Yet another oxygen (or nitrogen) at a similar to 2.6 angstrom distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a similar to 2.6 angstrom distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (∼5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(•). This can be followed by the induction of a split EPR signal from Y(Z)(•) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(•). Here we report on the pH dependence for the induction of S(2)'Y(Z)(•) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ∼ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ∼ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ∼ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(•).

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (

The Split Epr Signals From YZ • In Magnetic Interaction With The Camn4, Cluster Offer A Way To Probe For Oxidation Of YZ In Active Psii. We Have Studied How Ph (Ph 4.0–6.5) Affects The Formation Of The Split S0 Signal Induced By Low Temperature Illumination. Maximum Signal Intensity Was Observed Around Ph 6.3. The Signal Intensity Decreased With An Apparent PK,A Of 5.1. The Results Are Compared And Discussed In Context To Previousobservations At Room Temperature.

A split EPR signal can be induced at 5 K in Photosystem II in the S3-state by light in the range of 400–900 nm. To investigate if the same mechanism is involved in the signal induction in the full spectral range we compared the properties of the S3 signal induced by 830 nm light or white light. Our results indicate that the same mechanism is responsible for the formation of the “Split S3” signal in the whole spectral range. The mechanism of the “Split S3” signal is not P680 driven but is instead driven by manganese excitation.

ESE field sweep and two and three pulse ESEEM measurements were performed on the Split S1 signal induced by illumination at liquid He temperatures of the PSII centers poised in the S1-state. The field sweep experiments in the radical region indicated possible involvement of YZ• in the formation of the Split S1 EPR signal. Frequency domain ESEEM revealed light inducible features which origin is discussed with respect to the Split S1 signal formation.

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S(0), S(1), and S(3) states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S(1) --S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --S(3) transition following the second flash.

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ• and the Mn 4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0°C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S 3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ• and the Mn4Ca cluster. (Less)

Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.