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G W Brudvig

Publications and source records attributed to G W Brudvig.

At least 19 recordsLinked to original sources
PubMed1998

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z.

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.

Cell Division↗
PubMed1998

Catalase-free photosystem II: the O2-evolving complex does not dismutate hydrogen peroxide.

A photosystem II (PSII) membrane-associated heme catalase has been identified as a major source of the dark H2O2-dismutation reaction in PSII membrane samples [Sheptovitsky, Y. G., and Brudvig, G. W. (1996) Biochemistry 35, 16255-16263]. Based on this finding, a catalase-free PSII membrane sample was prepared by using mild heat treatment to deplete most of the PSII membrane-associated heme catalase followed by inhibition of the residual catalase with 50 mM 3-amino-1,2,4-triazole, a specific heme catalase inhibitor that binds covalently to compound I. After these treatments, the PSII membrane sample exhibited only 0.02% of the original H2O2-dismutation activity when assayed in the presence of 20 mM 3-amino-1,2,4-triazole. This small residual H2O2-dismutation activity is attributed to adventitious metal ions or the non-heme iron in PSII because the activity was still present in a Mn-depleted PSII sample but was completely suppressed by adding 5 mM ferricyanide to the assay buffer; the effect of ferricyanide is attributed to oxidation of H2O2-dismutating cations. Although the H2O2-dismutation activity was completely eliminated by these treatments, the light-induced O2-evolution activity was retained. A single saturating flash given to catalase-free PSII membranes did not induce any H2O2-dismutation activity. These results demonstrate that the S1/S-1 and S2/S0 cycles of the O2-evolving complex of PSII do not occur in the presence of H2O2, as proposed by Velthuys, B., and Kok, B. [(1978) Biochim. Biophys. Acta 502, 211-221]. The light-induced O2-evolution activity in catalase-free PSII was found to be irreversibly impaired by micromolar concentrations of H2O2. Thus, it is possible that the PSII membrane-associated heme catalase plays an important role in protection of the O2-evolving complex from damage by H2O2.

Acatalasia↗
PubMed1998

Calcium binding studies of photosystem II using a calcium-selective electrode.

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.

Binding Sites↗
PubMed1997

Fluorescence quenching by chlorophyll cations in photosystem II.

Although fluorescence is widely used to study photosynthetic systems, the mechanisms that affect the fluorescence in photosystem II (PSII) are not completely understood. The aim of this study is to define the low-temperature steady-state fluorescence quenching of redox-active centers that function on the electron donor side of PSII. The redox states of the electron donors and acceptors were systematically varied by using a combination of pretreatments and illumination to produce and trap, at low temperature, a specific charge-separated state. Electron paramagnetic resonance spectroscopy and fluorescence intensity measurements were carried out on the same samples to obtain a correlation between the redox state and the fluorescence. It was found that illumination of PSII at temperatures between 85 and 260 K induced a fluorescence quenching state in two phases. At 85 K, where the fast phase was most prominent, only one electron-transfer pathway is active on the donor side of PSII. This pathway involves electron donation to the primary electron donor in PSII, P680, from cytochrome b559 and a redox-active chlorophyll molecule, ChlZ. Oxidized ChlZ was found to be a potent quencher of chlorophyll fluorescence with 15% of oxidized ChlZ sufficient to quench 70% of the fluorescence intensity. This implies that neighboring PSII reaction centers are energetically connected, allowing oxidized ChlZ in a few centers to quench most of the fluorescence. The presence of a well-defined quencher in PSII may make it possible to study the connectivity between antenna systems in different sample preparations. The other redox-active components on the donor side of PSII studied were the O2-evolving complex, the redox-active tyrosines (YZ and YD), and cytochrome b559. No significant changes in fluorescence intensity could be attributed to changes in the redox state of these components. The fast phase of fluorescence quenching is attributed to the rapid photooxidation of ChlZ, and the slow phase is attributed to multiple turnovers providing for further oxidation of ChlZ and irreversible photoinhibition. Significant photoinhibition only occurred at Chl concentrations below 0.7 mg/mL and above 150 K. The reversible oxidation of ChlZ in intact systems may function as a photoprotection mechanism under high-light conditions and account for a portion of the nonphotochemical fluorescence quenching.

Cations↗
PubMed1997

The tetranuclear manganese cluster in photosystem II: location and magnetic properties of the S2 state as determined by saturation-recovery EPR spectroscopy.

The spin-lattice relaxation enhancement of the dark-stable tyrosine radical, YD., by the S2 state of the O2-evolving complex (OEC) of photosystem II (PSII) has been measured by using saturation-recovery EPR spectroscopy. Two forms of the S2 state have been compared: the multiline EPR signal species in untreated PSII and the altered multiline EPR signal species in NH3-treated PSII. Previous work has shown that the non-single-exponential spin-lattice relaxation kinetics of YD. in S2-state PSII result from a dipole-dipole interaction with the Mn4 cluster of the OEC. By taking into account the temperature variation of the effective magnetic moment of the S2-state multiline EPR signal form of the OEC, we provide a quantitative analysis of its temperature-dependent enhancement of the spin-lattice relaxation of YD.. Different spin states of the Mn4 cluster in the S2 state are responsible for the effect at different temperature regimes: for T </= 10 K, it is the ground spin state (S = 1/2); for T >/= 30 K, it is the first excited spin state; and at intermediate temperatures, the contributions of the two spin states are comparable. The relaxation enhancement of YD. is equivalent for both forms of the S2-state multiline EPR signal examined, indicating that the magnetic properties of the Mn4 cluster are very similar in the S2 state for both untreated and NH3-treated PSII. EPR progressive microwave-power saturation has also been used to assess the spin-lattice relaxation properties of the Mn4 cluster giving the altered S2-state multiline EPR signal in the NH3 derivative of PSII. The Orbach mechanism is shown to provide the dominant relaxation pathway; the energy difference between the ground and first excited spin states is estimated to be 30 +/- 2 cm-1, which is very similar to the value found for the S2-state multiline EPR signal species in untreated PSII. Below 4 K, the effectiveness of the S2-state multiline EPR signal species as a spin relaxation enhancer of YD. drops dramatically. This is interpreted to occur because of temperature-dependent 55Mn nuclear spin-lattice relaxation which causes averaging of the effective Larmor frequency of the S2-state multiline EPR signal species during the time scale for spin-lattice relaxation of YD.; because the line shape of the S2-state multiline EPR signal is dominated by isotropic 55Mn nuclear hyperfine splittings, such nuclear relaxation processes allow frequencies in near resonance with that of YD. to be accessed, thereby producing a greater relaxation enhancement. By using a dipolar model that includes the line shapes of both the YD. and S2-state multiline EPR signals, the spin-lattice relaxation enhancement of YD. is analyzed to obtain a lower limit of 22 A for the distance between YD. and the OEC. Together with recent studies showing a close proximity of the Mn4 cluster to YZ., these results provide further support for an asymmetric location of the Mn4 cluster with respect to the two redox-active tyrosines in PSII.

Ammonia↗
PubMed1996

Isolation and characterization of spinach photosystem II membrane-associated catalase and polyphenol oxidase.

Photosystem II (PSII) membranes exhibit catalase and polyphenol oxidase (PPO) activities. Mild heat treatment of PSII membranes for 90 min at 30 degrees C releases most of these enzyme activities into the supernatant, accompanied by a 7-fold activation of PPO. In contrast, mild heat treatment of thylakoid membranes does not release significant amounts of either activity, indicating that both enzymes are bound to the luminal surface of the thylakoid membrane. The heat-released PSII membrane-associated catalase and PPO have been purified and characterized. Catalase activity was correlated with a 63 kDa polypeptide which was purified by batch adsorption to anion-exchange beads followed by gel filtration. The PSII membrane-associated catalase is unstable in solution, probably due to irreversible aggregation. The enzyme was characterized in terms of molecular and subunit size, amino-acid composition, UV-visible absorption, heme content, pH optimum, inhibitor sensitivity, and K(m) value for H2O2. Its properties indicate that the PSII membrane-associated catalase is a luminal thylakoid membrane-bound heme enzyme that has not been identified previously. The residual catalase activity of PSII membranes after mild heat treatment is irreversibly inhibited with 3-amino-1,2,4-triazole, a specific inhibitor of heme catalases, without inhibition of O2-evolution activity. This result indicates that little, if any, of the catalase activity from PSII membranes in the dark is catalyzed by the O2-evolving center of PSII. PPO activity was correlated with a 48 kDa polypeptide. However, the 48 kDa polypeptide and another heat-released polypeptide of 72 kDa have the same N-terminal sequence, which is also identical to that of a known 64 kDa protein [Hind, G., Marshak, D. R., & Coughlan, S. J. (1995) Biochemistry 34, 8157-8164]. During heat treatment of PSII membranes and further manipulations it was found that the 72 kDa polypeptide was largely converted into the 48 kDa polypeptide. Thus, the 72 kDa polypeptide appears to be a latent precursor of the active 48 kDa PPO. The PSII membrane-associated PPO was purified by anion-exchange chromatography and was characterized in terms of substrate specificity, pH optimum, inhibitor sensitivity and native molecular weight. The heat-released PPO appears to be identical to the enzyme previously isolated from spinach thylakoid membranes [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].

Amino Acids↗
PubMed1996

Reversible binding of nitric oxide to tyrosyl radicals in photosystem II. Nitric oxide quenches formation of the S3 EPR signal species in acetate-inhibited photosystem II.

Continuous illumination at temperatures above 250 K of photosystem II samples which have been depleted of calcium or chloride or treated with fluoride, acetate, or ammonia results in production of a broad radical EPR signal centered at g = 2.0. This EPR signal, called the S3 EPR signal, has been attributed to an organic radical interacting with the S2 state of the oxygen-evolving complex to give the species S2X+ (X+ = organic radical). A tyrosine radical has been proposed as the species responsible for the S3 EPR signal. On the basis of experiments demonstrating that nitric oxide binds reversibly to the tyrosyl radical in ribonucleotide reductase, nitric oxide has been used to probe the S3 EPR signal in acetate-treated photosystem II. In experiments using manganese-depleted photosystem II, nitric oxide was found to bind reversibly to both redox-active tyrosines, YD* and YZ*, to form EPR-silent adducts. Next, acetate-treated photosystem II was illuminated to form the S3 EPR signal in the presence of nitric oxide to test whether the S3 EPR signal behaves like YZ*. Under conditions that produce the maximum yield of the S3 EPR signal in acetate-treated photosystem II, no S3 EPR signal was observed in the presence of nitric oxide. Upon removal of nitric oxide, the S3 EPR signal could be induced. Quenching of the S3 EPR signal by nitric oxide yielded an S2-state multiline EPR signal. Its amplitude was 45% of that found for uninhibited photosystem II illuminated at 200 K; this yield is the same as the yield of the S3 EPR signal under equivalent conditions but without nitric oxide. These results suggest that the S3 EPR signal is due to the configuration S2YZ* in which the S2 state of the oxygen-evolving complex gives a broadened multiline EPR signal as a result of exchange and dipolar interactions with YZ*. The binding of nitric oxide to YZ* to form a diamagnetic YZ-NO species uncouples the S2 state from YZ*, yielding a noninteracting S2-state multiline EPR signal species.

Acetates↗
PubMed1996

EPR spectroscopic characterization of neuronal NO synthase.

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exits as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 HZ at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH., was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D=5.2cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.

Animals↗
PubMed1996

Formation and decay of the S3 EPR signal species in acetate-inhibited photosystem II.

A 230-G-wide EPR signal is induced in acetate-treated photosystem II by 30 s of illumination at 277 K followed by freezing under illumination to 77 K [MacLachlan, D. J., & Nugent, J. H. A. (1993) Biochemistry 32, 9772-9780]. This signal, referred to as the S3 EPR signal, has been interpreted to arise from an S2X+ species where X+ is an amino acid radical. Investigation of the factors responsible for the formation and decay of the S3 EPR signal reveals that the yield of the S3 EPR signal is strongly temperature-dependent and depends on the rate of oxidation of QA-. Quantitation of the number of centers contributing to the S3 EPR signal produced by the optimal continuous illumination times of 3 min at 250 K, 30 s at 273 K, and 5 s at 294 K gave values of 13, 38, and 49 +/- 3%, respectively. By using 5 s of illumination at 294 K to induce the S3 EPR signal, and then illumination at 200 K to reduce QA, both the S3 and QA(-)Fe EPR signals were induced in high yield. This result indicates that the S3 EPR signal does not arise from an acceptor-side species. When saturating laser flashes were used to induce the S3 EPR signal in a dark-prepared, dark-adapted, acetate-treated sample, the yield was small after one flash and close to maximal after two flashes. An EPR signal at g = 4.1 was observed to be formed at intermediate times during the decay of the S3 EPR signal in the dark; the rates of decay of the S3 EPR signal at 273 and 294 K corresponded to the rates of formation of the g = 4.1 EPR signal. These results, together with the flash results, indicate that two steps are involved in both the generation and decay of the S3 EPR signal. The rates of formation and decay of both the S3 and QA(-)Fe EPR signals were measured at 250, 273, and 294 K. A kinetic model is presented that accounts for these kinetic data and the yield of the S3 EPR signal.

Acetates↗
PubMed1995

Spectroscopic evidence for the symmetric location of tyrosines D and Z in photosystem II.

Saturation-recovery EPR spectroscopy has been used to probe the location of the redox-active tyrosines, YD (tyrosine 160 of the D2 polypeptide, cyanobacterial numbering) and YZ (tyrosine 161 of the D1 polypeptide), relative to the non-heme Fe(II) in Mn-depleted photosystem II (PSII). Measurements have been made on PSII membranes isolated from spinach and on PSII core complexes purified from the cyanobacterium Synechocystis sp. PCC 6803. In the case of Synechocystis PSII, site-directed mutagenesis of the YD residue to either phenylalanine (Y160F) or methionine (Y160M) was done to eliminate the dark-stable YD.species and, thereby, allow direct spectroscopic observation of the YZ. EPR signal. The spin-lattice relaxation transients of both YD. and YZ. were non-single-exponential due to a dipolar interaction with one of the other paramagnetic species in PSII. Measurements on CN(-)-treated, Mn-depleted cyanobacterial PSII, in which the non-heme Fe(II) was converted into its low-spin, diamagnetic state, proved that the non-heme Fe(II) was the sole spin-lattice relaxation enhancer for both the YD. and YZ. radicals. This justified the use of a dipolar model in order to fit the saturation-recovery EPR data, which were taken over the temperature range 4-70 K. The dipolar rate constants extracted from the fits were identical in magnitude and had the same temperature dependence for both YD. and YZ.. The observation of identical dipolar interactions between YD. and YZ. and the non-heme Fe(II) shows that the distance from each tyrosine to the non-heme Fe(II) is the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyanobacteria↗
PubMed1994

Location of chlorophyllZ in photosystem II.

Saturation-recovery and progressive microwave power saturation EPR spectroscopies have been used to probe the location of the chlorophyllZ+ (ChlZ+) radical species in Mn-depleted photosystems II (PSII). The spin-lattice relaxation transients of ChlZ+ were non-single-exponential due to a dipole-dipole interaction with one of the other paramagnetic centers in PSII. Measurements on CN(-)-treated, Mn-depleted PSII membrane samples, in which the non-heme Fe(II) is converted into its low-spin, diamagnetic form, confirmed that the non-heme Fe(II) caused the dipolar relaxation enhancement of ChlZ+. The saturation-recovery EPR data were fit to a dipolar model [Hirsh, D. J., Beck, W. F., Innes, J. B., & Brudvig, G. W. (1992) Biochemistry 31, 532] which takes into account the isotropic (scalar) and orientation-dependent (dipolar) contributions to the spin-lattice relaxation of the radical. The temperature dependence of the dipolar rate constants of ChlZ+ was identical to the temperature dependencies recently observed for the stable tyrosine radical, YD., and the special pair bacteriochlorophyll radical, (BChla)2+, in PSII and in reaction centers from Rhodobacter sphaeroides, respectively. Because the non-heme Fe(II) is known to cause a dipolar relaxation enhancement of the radicals in both of the latter cases, this result provides further evidence that the non-heme Fe(II) causes the dipolar relaxation enhancement of ChlZ+ and, moreover, demonstrates that the magnetic properties of the non-heme Fe(II) in PSII and in reaction centers from Rhodobacter sphaeroides are very similar. By using the known Fe(II)-(BChla)2+ distance for calibration, we estimate the Fe(II)-ChlZ+ distance to be 39.5 +/- 2.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriochlorophylls↗
PubMed1993

Spectroscopic evidence from site-directed mutants of Synechocystis PCC6803 in favor of a close interaction between histidine 189 and redox-active tyrosine 160, both of polypeptide D2 of the photosystem II reaction center.

The reaction center of photosystem II of oxygenic photosynthesis contains two redox-active tyrosines called Z and D, each of which can act as an electron donor to the oxidized primary electron donor, P680+. These tyrosines are located in homologous positions on the third transmembrane alpha-helix of each of the two homologous polypeptides, D1 and D2, that comprise the reaction center. Tyrosine D of polypeptide D2 has been proposed, upon oxidation, to give up its phenolic proton to a nearby basic amino acid residue, forming a neutral radical. Modeling studies have pointed to His190 (spinach numbering) as a likely candidate for this basic residue. As a test of this hypothesis, we have constructed three site-directed mutations in the D2 polypeptide of the cyanobacterium Synechocystis sp. PCC6803. His189 (the Synechocystis homologue of His190 of spinach) has been replaced by glutamine, aspartate, or leucine. Instead of the normal D. EPR signal (g = 2.0046; line width 16-19 G), PSII core complexes isolated from these three mutants show an altered dark-stable EPR signal with a narrowed line width (11-13 G), and g values of 2.0046, 2.0043, and 2.0042 for the His189Gln, His189Asp, and His189Leu mutants, respectively. Despite the reduced line width, these EPR signals show g values and microwave-power saturation properties similar to the normal D. signal. Furthermore, specific deuteration in one of those mutants at the 3 and 5 positions of the phenol ring of the photosystem II reaction center tyrosines results in a loss of hyperfine structure of the EPR signal, proving that the signal indeed arises from tyrosine.2+ This observation provides support for a model in which an imidazole nitrogen of His189 accepts the phenolic proton of Tyr160 upon oxidation of D, forming a back hydrogen bond to the phenolic oxygen of the neutral tyrosyl radical.

Cyanobacteria↗
PubMed1992

Reevaluation of the stoichiometry of cytochrome b559 in photosystem II and thylakoid membranes.

The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.

Cytochrome b Group↗
PubMed1992

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome b Group↗
PubMed1992

Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II.

The stable tyrosine radical YD. (tyrosine 160 in the D2 polypeptide) in photosystem II (PSII) exhibits nonexponential electron spin-lattice relaxation transients at low temperature. As previously reported, the tetranuclear Mn complex in PSII significantly enhances the spin-lattice relaxation of YD.. However, in Mn-depleted PSII membranes, the spin-lattice relaxation transients of YD. are also nonexponential, and progressive power saturation (P 1/2) experiments show that it does not behave like an isolated tyrosine radical. A model is developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a random orientation in a magnetic field. This model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of three relaxation rate constants: the "intrinsic" relaxation rate, the relaxation rate due to scalar exchange, and the relaxation rate due to dipole-dipole interactions. The intrinsic and the scalar exchange relaxation rates are isotropic and together contribute a single rate constant to the spin-lattice relaxation transients. However, the dipolar relaxation rate is orientation dependent. Each orientation contributes a different dipolar relaxation rate constant to the net spin-lattice relaxation rate constant. The result is a superposition of single-exponential recoveries, each with a different net rate constant, causing the observed saturation-recovery transients to be non-(single)-exponential. Saturation-recovery relaxation transients of YD. are compared with those of a model tyrosine radical, generated by UV photolysis of L-tyrosine in a borate glass. From this comparison, we conclude that scalar exchange does not make a significant contribution to the spin-lattice relaxation of YD. in Mn-depleted PSII. We account for the nonexponential relaxation transients obtained from YD. in Mn-depleted PSII membranes in terms of dipolar-induced relaxation enhancement from the non-heme Fe(II). From simulations of the spin-lattice relaxation transients, we obtain the magnitude of the magnetic dipolar interaction between YD. and the non-heme Fe(II), which can be used to calculate the distance between them. Using data on the non-heme Fe(II) in the reaction center of Rhodobacter sphaeroides to model the non-heme Fe(II) in PSII, we calculate a YD.-Fe(II) distance of greater than or equal to 38 A in PSII. This agrees well with the distance predicted from the structure of the bacterial reaction center.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Mechanism of irreversible inhibition of O2 evolution in photosystem II by Tris(hydroxymethyl)aminomethane.

The dark reaction of tris(hydroxymethyl)aminomethane (Tris) with the O2-evolving center of photosystem II (PSII) in the S1 state causes irreversible inhibition of O2 evolution. Similar inhibition is observed for several other amines: NH3, CH3NH2, (CH3)2NH, ethanolamine, and 2-amino-2-ethyl-1,3-propanediol. In PSII membranes, both depleted of the 17- and 23-kDa polypeptides and undepleted, the rate of reaction of Tris depends inversely upon the Cl- concentration. However, the rate of reaction of Tris is about 2-fold greater with PSII membranes depleted of the 17- and 23-kDa polypeptides than with undepleted PSII membranes. We have used low-temperature electron paramagnetic resonance (EPR) spectroscopy to study the effect of Tris on the oxidation state of the Mn complex in the O2-evolving center, to monitor the electron-donation reactions in Tris-treated samples, and to observe any loss of the Mn complex (forming Mn2+ ions) after Tris treatment. We find that Tris treatment causes loss of electron-donation ability from the Mn complex at the same rate as inhibition of O2 evolution and that Mn2+ ions are released. We conclude that Tris reduces the Mn complex to labile Mn2+ ions, without generating any kinetically stable, partially reduced intermediates, and that the reaction occurs at the Cl(-)-sensitive site previously characterized in studies of the reversible inhibition of O2 evolution by amines.

Electron Spin Resonance Spectroscopy↗