EPR characterization of the CP47-D1-D2-cytochrome b-559 complex of photosystem II

A photosystem II complex consisting of a 47-kDa chlorophyll-binding protein (CP47), the reaction center proteins D1 and D2, and cytochrome b-559 was characterized. Trace amounts of plastoquinone were found, indicating that the primary acceptor quinone QA has been extracted during purification. However, in the presence of ferricyanide, an EPR signal with the characteristic line shape and g value of the tyrosine radicals associated with photosystem II could be photoaccumulated in the majority of the reaction centers; in the absence of ferricyanide, or under low-temperature illumination conditions, a 9.5-11-G wide signal with a Gaussian line shape was observed at g = 2.003. Neither signal is observed in D1-D2-b-559 complexes, indicating that retention of CP47 produces a more native, but quinone-depleted photosystem II reaction center. The tyrosine radical photogenerated at room temperature can be trapped at cryogenic temperatures; results are presented showing that this radical can arise from tyrosine YZ, from tyrosine YD, or from both species. Low-temperature EPR spectroscopy also revealed a pronounced split signal with contributions at g = 6.05 and g = 5.75, which is attributed to high-spin, non-heme Fe3+ with axial ligation symmetry which is probably the non-heme iron associated with the acceptor side of photosystem II.     

Dual-mode EPR study of new signals from the S3-state of oxygen-evolving complex in photosystem II

The light-induced new EPR signals at g = 12 and 8 were observed in photosystem II (PS II) membranes by parallel polarization EPR. The signals were generated after two flashes of illumination at room temperature, and the signal intensity had four flashes period oscillation, indicating that the signal origin could be ascribed to the S3-state. Successful simulations were obtained assuming S = 1 spin for the values of the zero-field parameters, D = +/-0.435 +/- 0. 005 cm-1 and E/D = -0.317 +/- 0.002. Orientation dependence of the g =12 and 8 signal intensities shows that the axial direction of the zero-field interaction of the manganese cluster is nearly parallel to the membrane normal.     

Electron paramagnetic resonance signals from the S(3) state of the oxygen-evolving complex. A broadened radical signal induced by low-temperature near-infrared light illumination

The tetranuclear manganese cluster responsible for the oxidation of water in photosystem II cycles through five redox states denoted S(i)() (i = 0, 1, 2, 3, 4). Progress has been made recently in the detection of weak low-field EPR absorptions in both the perpendicular and parallel modes, associated with the integer spin state S(3) [Matsukawa, T., Mino, H., Yoneda, D., and Kawamori, A. (1999) Biochemistry 38, 4072-4077]. We confirm observation of these signals and have obtained them in high yield by illumination of photosystem II membranes, in which the non-heme iron was chemically preoxidized. It is shown that a split g = 4 signal accompanies the S(3) state signals. The signals diminish in the presence of ethanol and vanish in the presence of methanol. This effect is similar to that exerted by these alcohols to the high-spin component (g = 4.1) of the S(2) state and suggests that the latter spin configuration is the precursor of the S(3) state low-field signals. The S(3) state shows similar sensitivity to infrared illumination as has been observed previously in the S(2) state [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. Illumination of the S(3) state with near-infrared light (700-900 nm), at temperatures around 50 K, results in the modification of the low-field signals and most notably to the appearance of a broad (DeltaH approximately 200 G) radical-type signal centered at g = 2. The signal is tentatively assigned to the interaction of the Mn cluster in a modified S(2) state with a radical.     

Probing subtle coordination changes in the iron-quinone complex of photosystem II during charge separation,by the use of NO

The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.     

Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with alpha-tocopherol quinone

Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using M?ssbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.     

Comparative study of the g=4.1 EPR signals in the S(2) state of photosystem II

The Mn(4) complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S(2) state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g5 (spin 5/2) and a signal at g=4.1 (or g=4.25). The g=4.1 signal could be generated under two distinct sets of conditions: either by illumination at room temperature or at 200 K in certain experimental conditions (g4(S) signal) or by near-infrared illumination between approximately 77 and approximately 160 K of the S(2)-multiline state (g4(IR) signal). The two g=4.1 signals arise from states which have quite different stability in terms of temperature. In the present work we have compared these two signals in order to test if they originate from the same or from different chemical origins. The microwave power saturation properties of the two signals measured at 4.2 K were found to be virtually identical. Their temperature dependencies measured at non-saturating powers were also identical. The presence of Curie law behavior for the g4(S) and g4(IR) signals indicates that the states responsible for both signals are ground states. The orientation dependence, anisotropy and resolved hyperfine structure of the two g4 signals were also found to be virtually indistinguishable. We have been unable to confirm the behavior reported earlier indicating that the g4(S) signal is an excited state, nor were we able to confirm the presence of signal from a higher excited state in samples containing the g4(S), nor a radical signal in samples containing the g4(IR). These findings are best interpreted assuming that the two signals have a common origin i.e. a spin 5/2 ground state arising from a magnetically coupled Mn-cluster of 4 Mn ions.     

Role of bicarbonate in photosystem II, the water-plastoquinone oxido-reductase of plant photosynthesis

Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II.

Molecular changes associated with the photoreduction of the primary quinone acceptor Qa of photosystem II have been characterized by Fourier transform infrared spectroscopy. This reaction was light-induced at room temperature on photosystem II membranes in the presence of hydroxylamine and diuron. A positive signal at 1478 cm-1 is assigned to the C---O stretching mode of the semiquinone anion, and can be correlated to the negative C=O mode(s) of the neutral QA at 1645 cm-1 and/or 16 cm-1. Analogies with bacterial reaction center are found in the amide I absorption range at 1672 cm-1, 1653 cm-1 and 1630 cm-1. The stabilization of QA- does not result from a large protein conformation change, but involves perturbations of several amino acid vibrations. At 1658 cm-1, a negative feature sensitive to 1H-2H exchange is tentatively assigned to a NH2 histidine mode, while tryptophan D2252 could contribute to the signal at 1560/1550 cm-1.     

Fe-S centers in lactyl-CoA dehydratase

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR study of the oxygen evolving complex in His-tagged photosystem II from the cyanobacterium Synechococcus elongatus

The Mn(4)-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S(0)-state (spin = 1/2) and the S(2)-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S(3)-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S(3)-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn(4)-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn(4)-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn(4)-cluster. Biochemical modifications (Sr(2+)/Ca(2+) substitution, acetate and NH(3) treatments) were also investigated. In Sr(2+)-reconstituted PSII, in addition to the expected modified S(2) multiline signal, a signal at g = 5.2 was present instead of the g approximately 4 signal seen in plant PSII. In NH(3)-treated samples, in addition to the expected modified S(2)-multiline signal, a g approximately 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximately 4 spectra arising from the Mn(4)-cluster in the S(2) state have not yet been published in cyanobacterial PSII. The detection of modified S(3)-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH(3)-treated PSII indicate that NH(3) is still bound in the S(3)-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.     

A reversible effect of low carbon monoxide concentrations on the EPR spectra of the periplasmic hydrogenase from Desulfovibrio vulgaris

The effect of low concentrations of CO (0.93 - 5.58 microM) on the EPR spectrum of the periplasmic non-heme iron hydrogenase from D. vulgaris has been investigated. The "g = 2.06" EPR signal is maximally induced (0.94 spin/molecule) at 46.5 microM CO and partial induction of the EPR signal could be observed at 0.93 microM CO. This effect is reversed by removal of the CO or irradiation of the hydrogenase with white light.     

The manganese site of the photosynthetic oxygen-evolving complex probed by EPR spectroscopy of oriented photosystem II membranes: the g = 4 and g = 2 multiline signals.

The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electron spin-polarized signal of the P800+A1(Q)- state in the homodimeric reaction center core complex of Heliobacterium modesticaldum

The function of menaquinone as electron acceptor A 1 was identified by EPR in the purified type 1 homodimeric reaction center core complex (RC core) of an anoxygenic photosynthetic bacterium, Heliobacterium modesticaldum. After illumination of the RC core at 210 K in the absence and presence of dithionite, we detected the radical of a special pair of bacteriochlorophyll g molecules (P800 (+)) at g = 2.0033 and a quinone-type radical at g = 2.0062, respectively, at 14 K. Flash excitation of the dark-frozen RC core at 14 K induced two types of transient EPR signals, i.e., the P800 (+) radical that decayed with a time constant of 3.7 ms and a much faster decay component that showed the electron spin polarization (ESP) pattern of E/A (E, emission; A, absorption). The latter one was assigned to the P800 (+)F X (-) radical pair state. A new ESP signal that had an apparent A/E/A/E pattern extended to the lower-magnetic-field side was transiently induced by the flash excitation in the RC core that was preilluminated at 210 K in the presence of ascorbate and subsequently cooled to 14 K in the light. The 210 K preillumination of the RC core in the presence of dithionite led to accumulation of the dark stable semiquinone-type signal at g = 2.0062 and increased the intensity of the light-induced P800 triplet signal. Flash excitation at 14 K induced the smaller A/E/A/E-type signal that had the greater contribution of the lower-magnetic-field envelope. This ESP signal could thus be ascribed to the P800 (+)A 1 (-) radical pair. The kinetics and spectral shape of this ESP signal suggest that menaquinone serves as secondary electron acceptor A 1 with the molecular orientation of its ring being somewhat different from that of phylloquinone in photosystem I.     

Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept

Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.     

Modeling of the D1/D2 proteins and cofactors of the photosystem II reaction center: implications for herbicide and bicarbonate binding.

A three-dimensional model of the photosystem II (PSII) reaction center from the cyanobacterium Synechocystis sp. PCC 6803 was generated based on homology with the anoxygenic purple bacterial photosynthetic reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis, for which the X-ray crystallographic structures are available. The model was constructed with an alignment of D1 and D2 sequences with the L and M subunits of the bacterial reaction center, respectively, and by using as a scaffold the structurally conserved regions (SCRs) from bacterial templates. The structurally variant regions were built using a novel sequence-specific approach of searching for the best-matched protein segments in the Protein Data Bank with the "basic local alignment search tool" (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990, J Mol Biol 215:403-410), and imposing the matching conformational preference on the corresponding D1 and D2 regions. The structure thus obtained was refined by energy minimization. The modeled D1 and D2 proteins contain five transmembrane alpha-helices each, with cofactors (4 chlorophylls, 2 pheophytins, 2 plastoquinones, and a non-heme iron) essential for PSII primary photochemistry embedded in them. A beta-carotene, considered important for PSII photoprotection, was also included in the model. Four different possible conformations of the primary electron donor P680 chlorophylls were proposed, one based on the homology with the bacterial template and the other three on existing experimental suggestions in literature. The P680 conformation based on homology was preferred because it has the lowest energy. Redox active tyrosine residues important for P680+ reduction as well as residues important for PSII cofactor binding were analyzed. Residues involved in interprotein interactions in the model were also identified. Herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modeled in the plastoquinone QB binding niche using the structural information available from a DCMU-binding bacterial reaction center. A bicarbonate anion, known to play a role in PSII, but not in anoxygenic photosynthetic bacteria, was modeled in the non-heme iron site, providing a bidentate ligand to the iron. By modifying the previous hypothesis of Blubaugh and Govindjee (1988, Photosyn Res 19:85-128), we modeled a second bicarbonate and a water molecule in the QB site and we proposed a hypothesis to explain the mechanism of QB protonation mediated by bicarbonate and water. The bicarbonate, stabilized by D1-R257, donates a proton to QB2- through the intermediate of D1-H252; and a water molecule donates another proton to QB2-. Based on the discovery of a "water transport channel" in the bacterial reaction center, an analogous channel for transporting water and bicarbonate is proposed in our PSII model. The putative channel appears to be primarily positively charged near QB and the non-heme iron, in contrast to the polarity distribution in the bacterial water transport channel. The constructed model has been found to be consistent with most existing data.     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex.

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.     

Photoreducible high spin iron electron paramagnetic resonance signals in dark-adapted Photosystem II: are they oxidised non-haem iron formed from interaction of oxygen with PSII electron acceptors?

An electron paramagnetic resonance (EPR) signal near g=6 in Photosystem II (PSII) membranes has been assigned to a high spin form of cytochrome (Cyt) b(559) (R. Fiege, U. Schreiber, G. Renger, W. Lubitz, V.A. Shuvalov, FEBS Lett. 377 (1995) 325-329). Here we have further investigated the origin of this signal. A slow formation of the signal during storage in the dark is observed in oxygen-evolving PSII membranes, which correlate with the oxidation of Fe(2+) by plastosemiquinone or oxygen. Removal of oxygen inhibits formation of the high spin iron signal. The g=6 EPR signal is photoreduced at cryogenic temperatures and is restored slowly by subsequent dark storage at 77 K. The amplitude of the photoreduced signal increases as the pH is lowered, which shows that the origin is not the hydroxyl ligated Cyt b(559) species proposed previously. Different cryoprotectants also influence the amplitude and lineshape of the high spin iron signal in a manner suggesting that smaller cryoprotectants can penetrate the iron environment. A correlation between the high spin iron and g=1.6 EPR signal assigned to an interaction involving the semiquinones of Qa and Qb is shown. It is concluded that the appearance of the high spin iron signal in oxygen-evolving PSII membranes involves reduced PSII electron acceptors and oxygen and suggests that the signal is from the non-haem iron of PSII.     

The g = 2 multiline EPR signal of the S2 state of the photosynthetic oxygen-evolving complex originates from a ground spin state.

The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.