Biomedical subjects

A W Rutherford

Publications and source records attributed to A W Rutherford.

At least 37 records · Page 2Linked to original sources

PubMed1998

Influence of herbicide binding on the redox potential of the quinone acceptor in photosystem II: relevance to photodamage and phytotoxicity.

Here we show that herbicide binding influences the redox potential (Em) of the plastoquinone QA/QA- redox couple in Photosystem II (PSII). Phenolic herbicides lower the Em by approximately 45 mV, while DCMU raises the Em by 50 mV. These shifts are reflected in changes in the peak temperature of thermoluminescence bands arising from the recombination of charge pairs involving QA-. The herbicide-induced changes in the Em of QA/QA- correlate with earlier work showing that phenolic herbicides increase the sensitivity of PSII to light, while DCMU protects against photodamage. This correlation is explained in terms of the following hypothesis which is based on reactions occurring in the bacterial reaction center. The back-reaction pathway for P680+QA- is assumed to be modulated by the free-energy gap between the P680+QA- and the P680+Ph- radical pairs. When this gap is small (i.e., when the Em of QA/QA- is lowered), a true back-reaction is favored in which P680+Ph- is formed, a state which decays forming a significant yield of P680 triplet. This triplet state of chlorophyll reacts with oxygen, forming singlet oxygen, a species likely to be responsible for photodamage. When the free-energy gap is increased (i.e., when the Em of QA/QA- is raised), the yield of the P680+Ph- is diminished and a greater proportion of the P680+QA- radical pair decays by an alternative, less damaging, route. We propose that at least some of the phytotoxic properties of phenolic herbicides may be explained by the fact that they render PSII ultrasensitive to light due to this mechanism.

Diuron↗

PubMed1998

Relationship between activity, D1 loss, and Mn binding in photoinhibition of photosystem II.

Photoinhibition of photosystem II (PSII) activity and loss of the D1 reaction center protein were studied in PSII-enriched membrane fragments in which the water-splitting complex was inhibited by depletion of either calcium or chloride or by removing manganese. The Ca2+-depleted PSII was found to be the least susceptible to inhibition by light as reported previously (Krieger, A., and Rutherford, A. W. (1997) Biochim. Biophys. Acta 1319, 91-98). This different susceptibility to light was not reflected in the extent of D1 protein loss. In Mn-depleted PSII the loss of activity and the loss of the D1 protein were correlated, while in Cl-- and Ca2+-depleted PSII, there was very little loss of the D1 protein. The production of free radicals and singlet oxygen was measured by EPR spin-trapping techniques in the different samples. 1O2 and carbon-centered radicals could be detected after photoinhibition of active PSII, while hydroxyl radical formation dominated in all of the other samples. In addition, photoinhibition of PSII was investigated in which the functional Mn cluster was reconstituted (i. e., photoactivated). As expected this led to a protection against photoinhibition. When the photoactivation procedure was done in the absence of Ca2+ no activity was obtained although a nonfunctional Mn cluster was formed. Despite the lack of activity the binding of Mn partially protected against the loss of D1. These data demonstrate that, during photoinhibition, the extent of D1 loss is neither affected by the water-splitting activity of the sample nor correlated to the kinetics of PSII activity loss. D1 loss seems to be independent of the chemical nature of the reactive oxygen species formed during photoinhibition and seems to occur only in the absence of Mn. It is proposed that Mn binding protects against D1 loss by maintaining a protein structure which is not accessible to cleavage.

Binding Sites↗

PubMed1998

The 18 kDa cytochrome c553 from Heliobacterium gestii: gene sequence and characterization of the mature protein.

The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii. After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa. The redox midpoint potential of the cytochrome c553 was determined to be +215 mV. The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238. The gx = trough could not be detected. In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion. The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids. The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry. Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety. The amino acid sequence deduced from the DNA sequence exhibits partial identity to the membrane-bound cytochrome c551 from Bacillus PS3 [Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochem. Biophys. Res. Commun. 1144, 213-219] and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri [Zumft, W. G., Braun, C., and Cuypers, H. (1994) Eur. J. Biochem. 219, 481-490].

Amino Acid Sequence↗

PubMed1998

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↗

PubMed1998

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↗

PubMed1998

Spin-lattice relaxation of the phyllosemiquinone radical of photosystem I.

The spin-lattice relaxation time (T1) of the phyllosemiquinone anion radical, A1-, of the photosystem I (PSI) reaction center, were measured between 4.5 and 85 K by electron spin-echo spectroscopy. The selective removal of the iron-sulfur centers, FA, FB, and FX, from PSI allowed the measurement of the intrinsic T1 of the A1- radical. The temperature dependence of the intrinsic (T1)-1 for A1- was found to be approximately T1.3 +/- 0.1. The spin-lattice relaxation of the reduced form of iron-sulfur center FX was also measured at low temperatures, in FA/FB-depleted PSI membranes. It was found that the fast-relaxing FX center enhances the spin-lattice relaxation of the phyllosemiquinone due to dipolar coupling. The effect of the reduced forms of FA/FB on the T1 of the phyllosemiquinone was minor compared to the effect of FX. By analyzing the data with a dipolar model in the light of limitations imposed by other information present in the literature, the distance between the phyllosemiquinone and FX in PSI is estimated to be 14.8 +/- 4 A.

Benzoquinones↗

PubMed1998

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.

Animals↗

PubMed1997

ESEEM study of the phyllosemiquinone radical A1.- in 14N- and 15N-labeled photosystem I.

The phyllosemiquinone radical of the photosystem I reaction center has been studied by electron spin echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of ESEEM data of the semiquinone in 14N- and 15N-labeled PSI and numerical simulations demonstrate the existence of two protein nitrogen nuclei coupled to the semiquinone. One of the 14N couplings is characterized by a quadrupolar coupling constant e2qQ/4h of 0.77 MHz, an asymmetry parameter eta of 0.18, and a hyperfine coupling tensor with an almost pure isotropic hyperfine coupling, i.e. (Axx, Ayy, Azz) = (1.3, 1.3, 1.5 MHz). The second nitrogen coupling is characterized by a quadrupolar coupling constant e2qQ/4h of 0.45 MHz, an asymmetry parameter eta of 0.85, and a weak hyperfine coupling tensor with a dominant anisotropic part, i.e. (Axx, Ayy, Azz) = (-0.2, -0.2, 1.5 MHz). On the basis of a comparison of the 14N-ESEEM data with 14N-NQR and 14N-ESEEM data from the literature, the first coupled nitrogen is assigned to the indole nitrogen of a tryptophan residue. The coupling of the second nitrogen is much weaker and therefore more difficult to assign. However, the simulated spectrum best describes an amino nitrogen of a histidine, although the amide group of an asparagine or glutamine cannot be ruled out. The possible origins of teh nitrogen hyperfine coupling are discussed in terms of the amino acid residues thought to be close to the semiquinone in PSI.

Benzoquinones↗

PubMed1997

Orientation of the phylloquinone electron acceptor anion radical in photosystem I.

The photosynthetic reaction center of photosystem I (PS I) contains a phylloquinone molecule (A1) which acts as a transient electron acceptor. In PS I form the cyanobacterium Synechocystis PCC 6803 under reducing conditions, we have photoaccumulated an EPR signal assigned to the phylloquinone radical anion. The phylloquinone EPR spectrum has been studied in oriented multilayers of PS I using EPR at 9 GHz. In addition, the phyllosemiquinone spectrum has been obtained at 283 GHz using high-field, high-frequency EPR spectroscopy. From the orientation dependence of the spectrum at 9 GHz and the resolved g values obtained at 283 GHz, the phyllosemiquinone ring plane was determined to be almost perpendicular to the membrane (76 degrees) while the oxygen-oxygen (O-O) axis of the quinone was found to make an approximate 63 degrees angle to the membrane plane. The orientation of the ring plane is similar to that determined for the quinone electron acceptor (QA) in the purple bacterial reaction center, while the orientation of the O-O axis is significantly different. The new orientation information, when taken with data in the literature, allows the position of the phylloquinone in the reaction center to be better defined.

Anions↗

PubMed1997

Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II.

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Amino Acid Sequence↗

PubMed1996

Membrane-bound c-type cytochromes in Heliobacillus mobilis. Characterisation by EPR and optical spectroscopy in membranes and detergent-solubilised material.

The spectral and electrochemical parameters, as well as the orientations of the heme plane with respect to the membrane plane, of the c-type hemes present in membrane fragments from Heliobacillus mobilis were characterised by optical and EPR spectroscopy. Cytochrome C53, was thereby shown to represent at least four and possibly five heme species with the following characteristics: Em = -60 mV +/- 10 mV, g, = 2.92, 60 degrees; Em = +90 mV +/- 10 mV, g, = 2.92, 90 degrees; Em = +120 mV +/- 20 mV, g, = 3.03; and Em = +170 mV +/- 20 mV, g, = 3.03. The latter component may correspond to two hemes with redox midpoint potentials of Em = +160 mV +/- 20 mV and Em = +180 mV +/- 20 mV (all Em values at pH 7.0). For the heme species having g, peaks at g approximately 3.03, determination of individual orientations was precluded due to the superposition of several differently oriented hemes. About one copy of each heme was found to be present per photosynthetic reaction centre, with the exception of the +120 mV component for which a stoichiometry of 2 hemes/reaction centre was obtained. The heme proteins were detergent-solubilised and partially purified. Three c-type cytochromes that migrated with apparent molecular masses of 18, 29 and 50 kDa were detected on SDS/PAGE. Optical redox titrations at pH 7.0 showed redox midpoint potentials of +160 mV +/- 10 mV for the 18-kDa cytochrome, and -60 mV +/- 10 mV, with possible contributions around +160 mV, for the 50-kDa cytochrome. A tentative attribution of heme species observed in membranes to the isolated heme proteins is presented. The results obtained on H. mobilis are compared with those reported for green sulphur bacteria.

Bacteria↗

PubMed1996

The role of the extrinsic 33 kDa protein in Ca2+ binding in photosystem II.

The role of the 33 kDa protein in Ca2+ binding was studied by comparing the EPR properties of photosystem II in the presence and absence of the 33 kDa protein and Ca2+. When the removal of the 33 kDa protein was carried out in the dark, a normal manganese multiline EPR signal could be observed when the S2 state was generated. In addition, the split S3 signal could not be generated by illumination at 273 K. Exposure of the 33 kDa protein-less photosystem II to room light did not lead to any change in the EPR properties of the S2 state, but the split S3 state signal at around g = 2 could then be generated, indicating that Ca2+ was released from this preparation during the exposure to light. Treatment of photosystem II lacking the 33 kDa protein with EGTA in the light led to a modification of the S2 state characterized by a dark-stable multiline EPR signal. Much lower EGTA concentrations were required in order to obtain this modification in the absence of the 33 kDa protein than was required when the 33 kDa protein was present. This indicates that the manganese cluster was more accessible to chelator binding when the 33 kDa protein was absent. When 33 kDa protein-less photosystem II was treated with EGTA in the dark, no modification of the multiline EPR signal of the S2 state of the manganese cluster occurred, nor was Ca2+ released as monitored by the inability to generate the split S3 signal. These chelator- and Ca(2+)-binding properties occurring in PSII lacking the 33 kDa protein are very similar to those observed previously in NaCl-washed PSII in which the 33 kDa protein is present (reviewed in Boussac & Rutherford, 1994a). It is concluded that the 33 kDa protein has little or no direct role in binding the Ca2+ ion which is required for oxygen evolution.

Bacterial Proteins↗

PubMed1996

Spin-lattice relaxation of the pheophytin, Pheo-, radical of photosystem II.

The spin-lattice relaxation times (T1) of the pheophytin anion radical, Pheo-, of the PSII reaction center, were measured between 5 and 80 K by electron spin-echo spectroscopy. The Pheo- was studied in Mn-depleted PSII reaction centers in which the primary quinone, QA, was doubly reduced. The selective conversion of the non-heme Fe2+ into its low-spin (S = O) state, in CN-treated PSII, allowed the measurement of the intrinsic T1 of the Pheo- radical. The temperature dependence of the intrinsic (T1)-1 was found to be approximately T1.3 +/- 0.1. In Mn-depleted PSII membranes the high-spin (S = 2) non-heme iron, enhances the spin-lattice relaxation of Pheo-. By analyzing the data with a dipolar model, the dipolar interaction (k1d) between the Pheo and the Fe2+ (S = 2) is estimated over the temperature range 5-80 K. Comparison with the dipolar coupling between the iron and the tyrosine, YD+, shows that the Pheo is much closer to the iron than the YD+ in the PSII reaction center. By scaling the reported Fe(2+)-YD+ distance by the ratio [k1dPheo-]/[k1dYD+], we estimate the Fe(2+)-Pheo- distance in PSII to be 20 +/- 4.2 A. This distance is close to the Fe(2+)-BPheo- distance in the bacterial reaction center, and this result provides further evidence that the acceptor sides of the reaction centers in PSII and bacteria are homologous.

Benzoquinones↗

PubMed1996

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↗

PubMed1996

Properties of the chloride-depleted oxygen-evolving complex of photosystem II studied by electron paramagnetic resonance.

The effects of different Cl- depletion treatments in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy and by measurements of oxygen-evolving activity. The results indicated that the oxygen-evolving complex of PS-II exhibits two distinct Cl(-)-dependent properties. (1) After Cl(-)-free washes at pH 6.3, a reversibly altered distribution of structural states of PS-II was observed, manifested as the appearance of a g = 4 EPR signal from the S2 state in a significant fraction of centers (20-40%) at the expense of the S2 multiline signal. In addition, small but significant changes in the shape of the S2 multiline EPR signal were observed. Reconstitution of Cl- to Cl(-)-free washed PS-II rapidly reversed the observed effects of the Cl(-)-free washing. The anions, SO4(2-) and F-, which are often used during Cl- depletion treatments, had no effect on the S2 EPR properties of PS-II under these conditions in the absence or presence of Cl-. Flash experiments and measurements of oxygen evolution versus light intensity indicated that the two structural states observed after the removal of Cl- at pH 6.3 originated from oxygen-evolving centers exhibiting a lowered quantum yield of water oxidation. (2) Depletion of Cl- in PS-II by pH 10 treatment reversibly inhibited the oxygen-evolving activity to approximately 15%. The pH 10 treatment depleted the Cl- from a site which is considered to be equivalent to that studied in most earlier work on Cl(-)-depleted PS-II. The S2 state in pH 10/Cl(-)-depleted PS-II was reversibly modified to a state from which no S2 multiline EPR signal was generated and which exhibited an intense S2 g = 4 EPR signal corresponding to at least 40% of the centers but possibly to a much larger fraction of centers. The state responsible for the intense S2 g = 4 signal generated under these conditions is unlike that observed after removal of Cl- from PS-II at pH 6.3, in that this state was more stable in the dark, showing a half-decay time of approximately 1.5 h at 0 degrees C, and was unable to undergo further charge accumulation. Nevertheless, a fraction of centers, probably different from those exhibiting the S2 g = 4 signal, was able to advance to the formal S3 state, giving rise to a narrow EPR signal around g = 2. Addition of the anions SO4(2-) or F- to pH 10/Cl(-)-depleted PS-II affected the properties of PS-II, resulting in EPR properties of the S2 state similar to those reported earlier following Cl- depletion treatment of PS-II in the presence of these anions. Surprisingly, after addition of F-, the g = 4 EPR signal showed a damped flash-dependent oscillation. In addition, a narrow signal around g = 2, corresponding to the formal S3 state, also showed a damped flash-dependent oscillation pattern. The presence of oscillating EPR signals (albeit damped) in F(-)-treated pH 10/Cl(-)-depleted PS-II indicates functional enzyme turnover. This was confirmed by measurements of the oxygen-evolving activity versus light intensity which indicated that in approximately 45% of oxygen-evolving centers the enzyme turnover was slowed by a factor of 2. The distinct Cl- depletion effects in PS-II observed under the two different Cl- depletion treatments are considered to reflect the presence of two distinct Cl(-)-binding sites in PS-II.

Binding Sites↗

PubMed1996

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II.

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.

Amino Acid Sequence↗

PubMed1995

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↗

PubMed1995

Charge recombination reactions in photosystem II. I. Yields, recombination pathways, and kinetics of the primary pair.

Recombination reactions of the primary radical pair in photosystem II (PS II) have been studied in the nanosecond to millisecond time scales by flash absorption spectroscopy. Samples in which the first quinone acceptor (QA) was in the semiquinone form (QA-) or in the doubly reduced state (presumably QAH2) were used. The redox state of QA and the long-lived triplet state of the primary electron donor chlorophyll (3P680) were monitored by EPR. The following results were obtained at cryogenic temperatures (around 20 K). (1) the primary radical pair, P680+Pheo-, is formed with a high yield irrespective of the redox state of QA. (2) The decay of the primary pair is faster with QA- than with QAH2 and could be described biexponentially with t1/2 approximately 20 ns (approximately 65%)/150 ns (approximately 35%) and t1/2 approximately 60 ns (approximately 35%)/250 ns (approximately 65%), respectively. The different kinetics may be due to electrostatic and/or magnetic effects of QA- on charge recombination or due to conformational changes caused by the double reduction treatment. (3) The yield of the triplet state 3P680 was high both with QA- and QAH2. (4) The triplet decay was much faster with QA- [t1/2 approximately 2 microseconds (approximately 50%)/20 microseconds (approximately 50%)] than with QAH2 [t1/2 approximately 1 ms (approximately 65%)/3 ms (approximately 35%)]. The short lifetime of the triplet with QA- explains why it was not detected earlier. The mechanism of triplet quenching in the presence of QA- is not understood; however it may represent a protective process in PS II. (5) Almost identical data were obtained for PS II-enriched membranes from spinach and PS II core preparations from Synechococcus. Room temperature optical studies were performed on the Synechococcus preparation. In samples containing sodium dithionite to form QA- in the dark, EPR controls showed that multiple excitation flashes given at room temperature led to a decrease of the QA-Fe2+ signal, indicating double reduction of QA. During the first few flashes, QA- was still present in the large majority of the centers. In this case, the yield of the primary pair at room temperature was around 50%, and its decay could be described monoexponentially with t1/2 approximately 8 ns (a slightly better fit was obtained with two exponentials: t1/2 approximately 4 ns (approximately 80%)/25 ns (approximately 20%).(ABSTRACT TRUNCATED AT 400 WORDS)

Cyanobacteria↗