Biochemistry. Water photolysis in biology.
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Calcium↗
Biomedical subjects
A Boussac
Publications and source records attributed to A Boussac.
Rapid formation of the stable tyrosyl radical in photosystem II.
Two symmetrically positioned redox active tyrosine residues are present in the photosystem II (PSII) reaction center. One of them, TyrZ, is oxidized in the ns-micros time scale by P680+ and reduced rapidly (micros to ms) by electrons from the Mn complex. The other one, TyrD, is stable in its oxidized form and seems to play no direct role in enzyme function. Here, we have studied electron donation from these tyrosines to the chlorophyll cation (P680+) in Mn-depleted PSII from plants and cyanobacteria. In particular, a mutant lacking TyrZ was used to investigate electron donation from TyrD. By using EPR and time-resolved absorption spectroscopy, we show that reduced TyrD is capable of donating an electron to P680+ with t1/2 approximately equal to 190 ns at pH 8.5 in approximately half of the centers. This rate is approximately 10(5) times faster than was previously thought and similar to the TyrZ donation rate in Mn-depleted wild-type PSII (pH 8.5). Some earlier arguments put forward to rationalize the supposedly slow electron donation from TyrD (compared with that from TyrZ) can be reassessed. At pH 6.5, TyrZ (t1/2 = 2-10 micros) donates much faster to P680+ than does TyrD (t1/2 > 150 micros). These different rates may reflect the different fates of the proton released from the respective tyrosines upon oxidation. The rapid rate of electron donation from TyrD requires at least partial localization of P680+ on the chlorophyll (PD2) that is located on the D2 side of the reaction center.
Chlorophyll↗
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.
Acetates↗
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.
Electron Spin Resonance Spectroscopy↗
A Two-Step Spin Crossover in
Measurement of magnetic susceptibility with temperature of a powdered iron(III) catecholate complex clearly indicated a two-step spin-crossover process S=(1/2)<-->S=5/2. The picture shows the plot of chi(M)T against temperature.
Journal Article↗
Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.
The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.
Cyanobacteria↗
Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.
The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.
Cytochrome c Group↗
Hydrogen bonding of redox-active tyrosine Z of photosystem II probed by FTIR difference spectroscopy.
The TyrZ./TyrZ FTIR difference spectrum is reported for the first time in Mn-depleted photosystem II (PS II)-enriched membranes of spinach, in PS II core complexes of Synechocystis sp. PCC 6803 WT, and in the mutant lacking TyrD (D2-Tyr160Phe). In Synechocystis, the v7'a(CO) and delta(COH) infrared modes of TyrZ are proposed to account at 1279 and 1255 cm-1. The frequency of these modes indicate that TyrZ is protonated at pH 6 and involved in a strong hydrogen bond to the side chain of a histidine, probably D1-His190. A positive signal at 1512 cm-1 is assigned to the v(CO) mode of TyrZ. on the basis of the 27 cm-1 downshift observed upon 13C-Tyr labeling at the Tyr ring C4 carbon. A second IR signal, at 1532 cm-1, is tentatively assigned to the v8a(CC) mode of TyrZ.. The frequency of the v(CO) mode of TyrZ. at 1512 cm-1 is comparable to that observed at 1513 cm-1 for the Tyr. obtained by UV photochemistry of tyrosinate in solution, while it is higher than that of TyrD. in WT PS II at 1503 cm-1 and that of non-hydrogen-bonded TyrD. in the D2-His189Gln mutant at 1497 cm-1 [Hienerwadel, R., Boussac, A., Breton, J., Diner, B. A., and Berthomieu, C. (1997) Biochemistry 36, 14712-14723]. This latter work and the present FTIR study suggest that hydrogen bonding induces an upshift of the v(CO) IR mode of tyrosyl radicals and that TyrZ. forms (a) stronger hydrogen bond(s) than TyrD. in WT PS II. Alternatively, the frequency difference between TyrZ. and TyrD. v(CO) modes could be explained by a more localized positive charge near the tyrosyl radical oxygen of TyrD. than TyrZ.. The TyrZ./TyrZ spectrum obtained in Mn-depleted PS II membranes of spinach shows large similarities with the S3'/S2' spectrum characteristic of radical formation in Mn-containing but Ca(2+)-depleted PS II, in support of the assignment using ESEEM of TyrZ. as being responsible for the split EPR signal observed upon illumination in these conditions [Tang, X.-S., Randall, D. W., Force, D. A., Diner, B. A., and Britt, R. D. (1996) J. Am. Chem. Soc. 118, 7638-7639]. The peak at 1514 cm-1 is assigned to the v(CO) mode of TyrZ. in these preparations, which indicates that Mn depletion only very slightly perturbs the immediate environment of TyrZ. phenoxyl.
Amino Acid Substitution↗
Effect of near-infrared light on the S2-state of the manganese complex of photosystem II from Synechococcus elongatus.
The Mn cluster of Photosystem II (PSII) from Synechococcus elongatus was studied using EPR. A signal with features between g = 5 and g = 9 is reported from the S2-state. The signal is attributed to the manganese cluster in a state with a spin 5/2 state. Spectral simulations of the signal indicate zero field splitting parameters where the |E/D| was 0.13. The new signal is formed by irradiating PSII samples which contain the spin = 1/2 S2-state using 813 nm light below 200 K. This effect is attributed to a spin-state change in the manganese cluster due to absorption of the IR light by the Mn-cluster itself. The signal is similar to that reported recently in PSII of plants [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. In plant PSII the comparable signal is formed at a lower temperature (optimally below 77 K), and gradual warming of the sample in the dark leads to the formation of the state responsible for the well-known g = 4.1 signal prior to formation of the spin 1/2 multiline signal. In the present work using cyanobacterial PSII, warming of the sample in the dark leads to the formation of the spin 1/2 multiline signal without formation of the g = 4 type signal as an intermediate. These observations provide a partial explanation for the long-standing "mystery of the missing g = 4 state" in cyanobacterial PSII. The observations are rationalized in terms of three possible states which can exist for S2: (i) the spin 1/2 multiline signal, (ii) the state responsible for the g = 4.1 signal, and (iii) the new spin 5/2 state. The relative stability of these states differs between plants and cyanobacteria.
Cyanobacteria↗
Effect of (13)C-, (18)O- and (2)H-labeling on the infrared modes of UV-induced phenoxyl radicals.
The structure and environment of redox active tyrosines present in several metalloenzymes can be studied by resonance Raman spectroscopy or Fourier transform infrared difference spectroscopy. Assignments of the vibrational modes in vivo often requires in vitro studies on model compounds. This approach is briefly reviewed. New results are shown on the influence of isotope-labeling on the infrared spectra of tyrosine, [Formula: see text] and phenol radicals obtained in vitro by UV-irradiation. The infrared spectra of the radicals are dominated by the [Formula: see text] mode at 1515-1504 cm(-1). The frequency shifts induced on this mode by (13)C- (2)H-, and (18)O-labeling are reported.
Journal Article↗
High-spin states (S >/= 5/2) of the photosystem II manganese complex.
The Mn4 complex which is involved in water oxidation in photosystem II (PSII) is known to exhibit two types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: either a multiline signal (S = 1/2) or a signal at g = 4.1 (S = 3/2 or S= 5/2). The S = 1/2 state can be converted to that responsible for the g = 4.1 signal upon the absorption of near-infrared (IR) light [Boussac, A., Girerd, J.-J., and Rutherford, A.W. (1996) Biochemistry 35, 6984-6989]. It is shown here that a third state gives rise to signals at g = 10 and 6. This state is formed by IR illumination of the S = 1/2 state at 65 K, a temperature where IR illumination leads to the loss of the S = 1/2 signal but to no formation of the g = 4.1 state. On the basis of the corresponding decrease of the S = 1/2 state, the new state can be trapped in approximately 40% of the PSII centers. Warming of the sample above 65 K, in the dark, leads to the loss of the g = 10 and 6 resonances with the corresponding appearance of the g = 4.1 signal. It is suggested that the IR-induced conversion of the S = 1/2 state into the g = 4.1 state at 150 K involves the transient formation of the new state. The new state is attributed to a S = 5/2 state of the Mn4 complex (although a S value > 5/2 is also a possibility). Spectral simulations indicate an E/D ratio of -0.05 with D </= 1 cm-1. The resonances at g = 10 and 6 correspond to the gz of the +/-5/2 and +/-3/2 transition, respectively. The temperature-dependent conversion of this S = 5/2 state into the g = 4.1 state is proposed to be due to relaxation of the ligand environment around the Mn4 cluster that leads to a change in the zero field splitting parameters, assuming an S = 5/2 value for the g = 4.1 state. The new form of the S2 state reported here may explain some earlier data where the S2 state was present and yet not detectable as either a S = 1/2 or a g = 4.1 EPR signal.
Chlorophyll↗
Metal chelating properties of adenylate kinase from Paracoccus denitrificans.
Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.
Adenylate Kinase↗
Fourier transform infrared difference spectroscopy of photosystem II tyrosine D using site-directed mutagenesis and specific isotope labeling.
Tyrosine D (TyrD), a side path electron carrier of photosystem II (PS II), has been studied by light-induced Fourier transform infrared (FTIR) difference spectroscopy in PS II core complexes of Synechocystis sp. PCC 6803 using the experimental conditions previously optimized to generate the pure TyrD./TyrD FTIR difference spectrum in PS II-enriched membranes of spinach [Hienerwadel, R., Boussac, A., Breton, J., and Berthomieu, C. (1996) Biochemistry 35, 115447-115460]. IR modes of TyrD and TyrD. have been identified by specific 2H- or 13C-labeling of the tyrosine side chains. The v8a(CC) and v19(CC) IR modes of TyrD are identified at 1615 and 1513-1510 cm-1, respectively. These frequencies show that TyrD is protonated. Comparison of isotope-sensitive signals in situ with those of the model compound p-methylphenol dissolved in different solvents leads to the assignment of the v7'a(CO) and delta(COH) modes of TyrD at 1275 and 1250 cm-1, respectively. It is shown that these modes and in particular the delta(COH) IR mode are very sensitive to the formation of hydrogen-bonded complexes with amide C=O or with imidazole nitrogen atoms. The frequencies observed in situ show that TyrD is hydrogen-bonded to the imidazole ring of a neutral histidine. For the radical TyrD., isotope-sensitive IR modes are identified at 1532 and 1503 cm-1. The signal at 1503 cm-1 is assigned to the v(CO) mode of TyrD. since it is sensitive to 13C-labeling at the ring carbon involved in the C4-O bond. The perturbation of TyrD and TyrD. IR modes upon site-directed replacement of D2-His189 by Gln confirms that a hydrogen bond exists between both TyrD and TyrD. and D2-His189. In the D2-His189Gln mutant, the v7'a(CO) mode of TyrD at 1267 cm-1 and the delta(COH) mode at approximately 1228 cm-1 show that a hydrogen bond is formed between TyrD and an amide carbonyl, probably that of the D2-Gln189 side chain. Electron nuclear double resonance (ENDOR) measurements have shown that TyrD. is hydrogen-bonded in the wild type but not in the mutant [Tang, X.-S., Chrisholm, D. A., Dismukes, G. C., Brudwig, G. W., and Diner, B. A. (1993) Biochemistry 32, 13742-13748]. The v(CO) mode of TyrD. at 1497 cm-1 is downshifted by 6 cm-1 compared to WT PS II, indicating that hydrogen bonding induces a frequency upshift of the v(CO) IR mode of Tyr.. IR signals from the Gln side chain v(C=O) mode are proposed to contribute at 1659 and 1692 cm-1 in the TyrD and TyrD. states, respectively. These frequencies are consistent with the rupture of a hydrogen bond upon TyrD. formation in the mutant. The frequency of the v(CO) mode of TyrD., observed at 1503 cm-1 for WT PS II, is intermediate between that observed at 1497 cm-1 in the D2-His189Gln mutant and at 1513 cm-1 for Tyr. formed by UV irradiation in borate buffer, suggesting weaker or fewer hydrogen bonds for TyrD. in PS II than in solution. The role of D2-His189 in proton uptake upon TyrD. formation is also investigated.
Bacterial Proteins↗
A new non-heme iron environment in Paracoccus denitrificans adenylate kinase studied by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy.
Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.
Adenylate Kinase↗
Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups.
Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2- groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins.
Bacteriorhodopsins↗
Fourier transform infrared difference study of tyrosineD oxidation and plastoquinone QA reduction in photosystem II.
Two redox active tyrosines are present in the homologous polypeptides D1 and D2 of photo-system II (PS II). TyrZ (D1-161) is involved in the electron transfer reactions resulting in oxygen evolution, while TyrD (D2-160) usually forms a dark-stable radical. In Mn-depleted PS II, TyrD. can be slowly reduced by exogenous reductants. Charge separation then results in the oxidation of TyrD and TyrZ and the reduction of the primary electron acceptor QA. The semiquinone QA- can be reoxidized by oxidants like ferricyanide. In the present work, experimental conditions leading to the generation of pure QA-/QA or TyrD./TyrD FTIR difference spectra have been optimized. Therefore, single-turnover flashes or short illuminations were performed on PS II samples in the presence of exogenous reductants or oxidants. The QA- and TyrD. radicals were generated with high yield and with a lifetime of several seconds or minutes allowing averaging of FTIR difference spectra with high signal to noise ratio. Both QA- formation and contributions at the electron donor side of PS II were monitored by EPR spectroscopy. In PS II samples at pH 6 in the presence of PMS, NH2OH, and DCMU, EPR measurements show that QA- is formed with high yield upon a 1 s illumination at 10 degrees C, while no radical from the electron donor side of PS II is detected. Therefore the QA-/QA FTIR spectrum obtained in these conditions shows only vibrational changes due to QA reduction in PS II. In contrast, a similar spectrum was recently interpreted in terms of dominant contributions from Chl+/Chl signals [MacDonald, G. M., Steenhuis, J. J., & Barry, B. A. (1995) J. Biol. Chem. 270, 8420-8428], although the contribution from the electron acceptor QA was not quantified. In particular, it is shown here that the large positive signal at 1478 cm-1 is due to the QA- state and not to a Chl+ mode. This band is not downshifted upon 15N-labeling of spinach PS II membranes within the +/- 1 cm-1 accuracy of the method and is therefore tentatively assigned to the v(C[symbol: see text]O) mode of the plastosemiquinone QA-. Also unchanged upon 15N-labeling, signals at 1644 and/or 1630 cm-1 are possible candidates for the v(C = O) mode(s) of neutral QA in PS II. The TyrD./TyrD FTIR spectrum is recorded at 4 degrees C on Tris-washed PS II membranes from spinach at pH 6 in the presence of phosphate, formate, and ferricyanide. EPR experiments performed on these samples show that almost all TyrD. is formed upon a 1 s illumination at 4 degrees C and that TyrD. is then reduced within 12 min in the dark. No contributions from TyrZ. or QA- are detected 2 s after illumination. It is thus possible to optimize experimental conditions to record the FTIR difference spectrum only due to TyrD photooxidation in PS II-enriched membranes of spinach. The TyrD./TyrD FTIR spectrum is compared to a cresol./cresol FTIR difference spectrum obtained by UV irradiation at 10 K of cresol at pH 8. The spectral analogies observed between the in vivo and in vitro spectra recorded either in H2O or in D2O suggest that IR modes of TyrD contribute at 1513 and 1252 cm-1. These frequencies are characteristic of a protonated tyrosine. A positive signal is observed at 1506 cm-1 for cresol. and at 1504 cm-1 for the TyrD. state. This suggests contribution of the TyrD. side chain at 1504 cm-1. A band at 1473 cm-1 was previously assigned to the v(CO) mode of TyrD. [MacDonald, G. M., Bixby, K. A., & Barry, B. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028]. In contrast, no positive signal is observed at 1473 cm-1 in the TyrD./TyrD FTIR difference spectrum presented here. The TyrD./TyrD spectrum also shows vibrational changes from peptide groups and amino acid side chains which are modified upon TyrD. formation. Proton release at the PS II protein surface upon TyrD. formation is deduced from differential signals at the v(PO) modes of phosphate.
Electron Spin Resonance Spectroscopy↗
Conversion of the spin state of the manganese complex in photosystem II induced by near-infrared light.
The manganese complex (Mn4) which is responsible for water oxidation in photosystem II is EPR detectable in the S2 state, one of the five redox states of the enzyme cycle. The S2 state is observable at 10 K either as a multiline signal (spin 1/2) or as a signal at g = 4.1 (spin 3/2 or spin 5/2). It is shown here that at around 150 K the state responsible for the multiline signal is converted to that responsible for the g = 4.1 signal upon the absorption of infrared light. This conversion is fully reversible at 200 K. The action spectrum of this conversion has its maximum at 820 nm (12 200 cm-1) and is similar to the intervalence charge transfer band in di-mu-oxo-(MnIIIMnIV) model systems. It is suggested that the conversion of the multiline signal to the g = 4.1 signal results from absorption of infrared light by the Mn cluster itself, resulting in electron transfer from MnIII to MnIV. The g = 4.1 signal is thus proposed to arise from a state which differs from that which gives rise to the multiline signal only in terms of this change in its valence distribution. The near-infrared light effect was observed in the S2 state of Sr(2+)-reconstituted photosystem II and in Ca(2+)-depleted, EGTA (or citrate-)-treated photosystem II but not in ammonia-treated photosystem II. Earlier results in the literature which showed that the g = 4.1 state was preferentially formed by illumination at 130 K are reinterpreted as being the result of two photochemical events: the first being photosynthetic charge separation resulting in an S2 state which gives rise to the multiline signal and the second being the conversion of this state to the g = 4.1 state due to the simultaneous and inadvertent presence of 820 nm light in the broad-band illumination given. There is therefore no reason to consider the state responsible for the g = 4.1 signal as a precursor of that which gives rise to the multiline signal.
Ammonia↗
ESEEM study of the plastoquinone anion radical (QA.--) in 14N- and 15N-labeled photosystem II treated with CN.
The nonheme iron of the photosystem II reaction center was converted to its low-spin state (S = 0) by treatment with CN-. This allowed the study of the plastoquinone, QA- anion radical by electron spin-echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of the ESEEM data of QA- in 14N- and 15N-labeled PSII demonstrates the existence of a protein nitrogen nucleus coupled to the QA-. The 14N coupling is characterized by a quadrupolar coupling constant e2qQ/4h = 0.82 MHz, an asymmetry parameter eta = 0.45, and hyperfine coupling constant A approximately 2.1 MHz. The 15N hyperfine coupling is characterized by T = 0.41 MHz and alpha iso approximately 3.3 MHz. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acids thought to be close to the QA- in PSII. Based on a comparison of the 14N ESEEM with 14N-NQR and 14N-ESEEM data from the literature, the most likely candidate is the amide nitrogen of the peptide backbone of Ala261 of the polypeptide D2, although the indole nitrogen of Trp254 and the imino nitrogen of His215 of D2 also remain candidates.
Cyanides↗