Characterization of the interaction between manganese and tyrosine Z in acetate-inhibited photosystem II

When acetate-inhibited photosystem II (PSII) membranes are illuminated at temperatures above 250 K and quickly cooled to 77 K, a 240 G-wide electron paramagnetic resonance (EPR) signal is observed at 10 K. This EPR signal arises from a reciprocal interaction between the spin 1/2 ground state of the S2 state of the Mn4 cluster, for which a multiline EPR signal with shifted 55Mn hyperfine peaks is observed, and the oxidized tyrosine residue, YZ*, for which a broadened YZ* EPR spectrum is observed. The S2YZ* EPR signal in acetate-inhibited PSII is the first in which characteristic spectral features from both paramagnets can be observed. The observation of distinct EPR signals from each of the paramagnets together with the lack of a half-field EPR transition indicates that the exchange and dipolar couplings are weak. Below 20 K, the S2YZ* EPR signal in acetate-inhibited PSII is in the static limit. Above 20 K, the line width narrows dramatically as the broad low-temperature S2YZ* EPR signal is converted to a narrow YZ* EPR signal at room temperature. The line width narrowing is interpreted to be due to averaging of the exchange and dipolar interactions between YZ* and the S2 state of the Mn4 cluster by rapid spin-lattice relaxation of the Mn4 cluster as the temperature is increased. Decay of the S2YZ* intermediate at 200 K shows that the g = 4.1 form of the S2 state is formed and that a noninteracting S2-state multiline EPR signal is not observed as an intermediate in the decay. This result shows that a change in the redox state of YZ induces a spin-state change in the Mn4 cluster in acetate-inhibited PSII. The interconversion between spin states of the Mn4 cluster in acetate-inhibited PSII supports the idea that YZ oxidation or YZ* reduction is communicated to the Mn4 cluster through a direct hydrogen-bonding pathway, possibly involving a ligand bound to the Mn4 cluster.     

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.     

Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 1. Instability or inefficient assembly of the manganese cluster in vivo

Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.     

The 23 and 17 kDa extrinsic proteins of photosystem II modulate the magnetic properties of the S1-state manganese cluster

An S1-state parallel polarization "multiline" EPR signal arising from the oxygen-evolving complex has been detected in spinach (PSII) membrane and core preparations depleted of the 23 and 17 kDa extrinsic polypeptides, but retaining the 33 kDa extrinsic protein. This S1-state multiline signal, with an effective g value of 12 and at least 18 hyperfine lines, has previously been detected only in PSII preparations from the cyanobacterium sp. Synechocystis sp. PCC6803 [Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J., and Britt, R. D. (1998) J. Am. Chem. Soc. 120, 447-448]. It is absent in PSII spinach membrane and core preparations that either fully retain or completely lack the 33, 23, and 17 kDa extrinsic proteins. The S1-state multiline signal detected in spinach PSII cores and membranes has the same effective g value and hyperfine spacing as the signal detected in Synechocystis PSII particles. This signal provides direct evidence for the influence of the extrinsic PSII proteins on the magnetic properties of the Mn cluster.     

Simulation of S2-state multiline EPR signal in oriented photosystem II membranes: structural implications for the manganese cluster in an oxygen-evolving complex

High-quality angular-dependent spectra of multiline electron paramagnetic resonance (EPR) signals from the S2-state Mn cluster in a photosynthetic oxygen-evolving complex (OEC) were obtained for partially oriented photosystem (PS) II membranes, and the magnetic structure of the Mn cluster has been studied by simulation analysis. The angular-dependent multiline spectra were simulated by taking into account the anisotropic properties of both hyperfine tensors of intrinsic Mn ions and g-tensor of the cluster in a tetranuclear model. The best-fit parameters for the simulation indicate that (a) the oxidation state of the S2-state Mn cluster is Mn(III, IV, IV, IV), (b) the electronic orbital configuration of the Mn(III) ion is (dpi)3[dz2(sigma))]1, (c) the effective g-tensor of the Mn cluster and the hyperfine tensor of the Mn(III) ion are axially symmetric, and their principal z-axes are nearly collinear each other, and (d) the z-axis of the dz2 orbital of the Mn(III) ion and the normal of the thylakoid membrane are at an angle of 50.1 +/- 1.8 degrees. The results are compatible with those of the oriented XAFS study [Mukerji, I., et al. (1994) Biochemistry 33, 9712-9721], and indicate that the O-O vector of the putative di-mu-oxo bridged Mn(III)-Mn(IV) dimer unit in the Mn cluster tilts by 43-56 degrees with respect to the normal of thylakoid membrane. A model of the arrangement of the di-mu-oxo bridged Mn(III)-Mn(IV) unit with respect to the thylakoid membrane is proposed.     

Synthesis and stability of isotopically labeled p-chloro-m-xylenol (PCMX)

The synthesis, reaction kinetics, and pH stability of isotopically labeled p-chloro-m-xylenol (PCMX) were evaluated. While base catalysis was more rapid than acid catalysis, the latter allowed the use of a cosolvent for deuterium and tritium labeling using as little as 250 microL labeled water. Both acid and base catalysis were markedly more rapid than that reported previously for the deuteration of PCMX and related phenols. Isotopic labeling occurred only at the 2 and 6 ring positions, ortho to the phenolic group of PCMX. No deuterium loss was observed after storage for 21 days at 37 degrees C over a pH range of 2-14. Isotopic loss was observed only below pH 2. The prepared 3H-labeled PCMX had a specific activity of 1.18 mCi/mmol, a radiochemical purity of 99.0%, and a chemical purity exceeding 99.0%, with a high stability during prolonged cold storage.     

Correlation between structure and magnetic spin state of the manganese cluster in the oxygen-evolving complex of photosystem II in the S2 state: determination by X-ray absorption spectroscopy

The structure of the manganese cluster in the S2 state with the g approximately 4 EPR signal (S2-g4 state) generated by 130 K illumination of photosystem II (PSII) membranes prepared from spinach has been investigated by X-ray absorption spectroscopy. The Mn X-ray absorption K-edge spectra of the S2-g4 state not only show a shift of the inflection point to higher energy from the S1 state but also reveal a different edge shape from that of the S2 state with the multiline signal (S2-MLS state). Extended X-ray absorption fine structure (EXAFS) studies of the Mn K-edge show that the structure of the Mn cluster in the S2-g4 state is distinctly different from those in the S2-MLS or S1 states. In the S2-g4 state, the second shell of back-scatters from the Mn absorber is found to contain two Mn-Mn distances of 2.73 and 2.85 A. We interpret this to indicate the presence of two nonequivalent di-mu-oxo-bridged Mn binuclear structures in the Mn cluster of the S2-g4 state. The third shell of the S2-g4 state at about 3.3 A also contains increased heterogeneity. By contrast, very little distance disorder was found to exist in the second shell of the S1 or S2-MLS states. A mechanism is proposed to explain these results in the context of our model for the Mn cluster and the EPR properties of the Mn complex in the S2 state.     

Coupled activation of the donor and the acceptor side of photosystem II during photoactivation of the oxygen evolving cluster

Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the QB site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between QA- and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from QA- to the QB site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of QB to its site, was found to precede the building of the Mn cluster. We also showed that TyrD was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of TyrD in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, TyrD, an auxiliary electron donor to P680+/TyrZ*, is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage.     

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.     

A Mn(II)-Mn(III) EPR signal arises from the interaction of NO with the S1 state of the water-oxidizing complex of photosystem II

It was shown recently [Goussias, C., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261-9266] that incubation of photosystem II preparations with NO at -30 degrees C in the dark results in the formation of a new intermediate of the water-oxidizing complex. This is characterized by an EPR signal centered at g = 2 with prominent manganese hyperfine structure. We have examined the detailed structure of the signal using difference EPR spectroscopy. This is facilitated by the observations that NO can be completely removed without decrease or modification of the signal, and illumination at 0 degree C eliminates the signal. The signal spans 1600 G and is characterized by sharp hyperfine structure. 14NO and 15NO cw EPR combined with pulsed ENDOR and ESEEM studies show no detectable contributions of the nitrogen nucleus to the spectrum. The spectrum bears similarities to the experimental spectrum of the Mn(II)-Mn(III) catalase [Zheng, M., Khangulov, S. V., Dismukes, G. C., and Barynin, V. V. (1994) Inorg. Chem. 33, 382-387]. Simulations allowing small variations in the catalase-tensor values result in an almost accurate reproduction of the NO-induced signal. This presents strong evidence for the assignment of the latter to a magnetically isolated Mn(II)-Mn(III) dimer. Since the starting oxidation states of Mn are higher than II, we deduce that NO acts effectively as a reductant, e.g., Mn(III)-Mn(III) + NO--> Mn(II)-Mn(III) + NO+. The temperature dependence of the nonsaturated EPR-signal intensity in the range 2-20 K indicates that the signal results from a ground state. The cw microwave power saturation data in the range 4-8 K can be interpreted assuming an Orbach relaxation mechanism with an excited state at delta = 42 K. Assuming antiferromagnetic coupling, -2JS1.S2, between the two manganese ions, J is estimated to be 10 cm-1. The finding that an EPR signal from the Mn cluster of PSII can be clearly assigned to a magnetically isolated Mn(II)-Mn(III) dimer bears important consequences in interpreting the structure of the Mn cluster. Although the signal is not currently assigned to a particular S state, it arises from a state lower than S1, possibly lower than S0, too.     

Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.     

Three-dimensional electron microscopy of the photosystem II core complex

Risk factors for central venous catheter (CVC)-related bacteraemia among infants admitted to a neonatal intensive care unit (NICU) were analysed and the impact of surveillance and continuing education on the incidence of this complication investigated. Among patients admitted to a NICU, CVC-related bacteraemia increased from 1/15 (7%) in 1987 to 11/26 (42%) in 1988 (P = 0.01). Coagulase-negative staphylococci isolated from bacteraemia patients showed clonal diversity by plasmid and chromosomal fingerprinting. A review of CVC care procedures suggested breaches in aseptic techniques. Catheter-care technique was revised to ensure maximal aseptic precautions, including the use of sterile gloves, gown and drapes. The new policy was promoted by a continuing education programme and regular feed-back of CVC-related bacteraemia incidence to NICU staff. In the four-year follow-up period, the attack-rate of CVC-related bacteraemia decreased to 18/156 (12%) patients [relative risk (RR): 0.27, 95% confidence interval (CI); 0.15-0.51; P < 0.001 vs the previous period]. By using the Cox's model proportional hazards, very low birthweight and the period before use of strict aseptic CVC care were found to be predictors of increased risk of catheter-related bacteraemia after adjustment for duration of catheterization. These data provide further evidence that strict aseptic precautions during the maintenance and utilization of CVC can contribute to lower the risk of catheter infection in critically ill neonates. Regular feedback of surveillance data was associated with a progressive decrease in incidence of infection, suggesting that it improved staff compliance with aseptic precautions.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Calcium induces binding and formation of a spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of photosystem II as precursor to the functional tetra-Mn/Ca cluster

Two new intermediates are described which form in the dark as precursors to the light-induced assembly of the photosynthetic water oxidation complex (WOC) from the inorganic components. Mn2+ binds to the apo-WOC-PSII protein in the absence of calcium at a high-affinity site. By using a hydrophobic chelator to remove Mn2+ and Ca2+ from the WOC and nonspecific Fe3+, a new EPR signal becomes visible upon binding of Mn2+ to this site, characterized by six-line 55Mn hyperfine structure (DeltaHpp = 96 +/- 1 G) and effective g = 8.3. These features indicate a high-spin electronic ground state (S = 5/2) for Mn2+ and a strong ligand field with large anisotropy. This signal is eliminated if excess Ca2+ or Mg2+ is present. A second Mn2+ EPR signal forms in place of this signal upon addition of Ca2+ in the dark. The yield of this Ca-induced Mn signal is optimum at a ratio of 2 Mn/PSII, and saturates with increasing [Ca2+] >/= 8 mM, exhibiting a calcium dissociation constant of KD = 1.4 mM. The EPR signal of the Ca-induced Mn center at 25 K is asymmetric with major g value of approximately 2.04 (DeltaHpp = 380 G) and a shoulder near g approximately 3.1. It also exhibits resolved 55Mn hyperfine splitting with separation DeltaHpp = 42-45 G. These spectral features are diagnostic of a variety of weakly interacting Mn2(II, II) pairs with electronic spins that are magnetic dipolar coupled in the range of intermanganese separations 4.1 +/- 0.4 A, and commonly associated with one or two carboxylate bridges. The calcium requirement for induction of the Mn2(II,II) signal matches the value observed for steady-state O2 evolution (Michaelis constant, KM approximately 1.4 mM), and for light-induced assembly of the WOC by photoactivation. The Ca-induced Mn2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, TyrZ+, than is the mononuclear Mn center present in the absence of Ca2+. The Ca-induced Mn2(II,II) signal serves as a precursor for photoactivation of the functional WOC and is abolished by the presence of Mg2+. Formation of the Mn2(II,II) EPR signal by addition of Ca2+ correlates with reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or reactivity of the Mn2(II,II) core with H2O2. We propose that calcium organizes the binding site for Mn ions in the apo-WOC protein and may even interact directly with the Mn2(II,II) pair via solvent or protein-derived bridging ligands.     

Use of a novel histidyl modifier to probe for residues on Tris-treated photosystem II membrane fragments that may bind functional manganese

In this paper, we investigate the effects of histidyl amino acid modification on high-affinity Mn binding to photosystem II (PSII) using methods similar to those used in the preceding paper [Ghirardi et al. (1998) Biochemistry 37, 0000] for carboxyl amino acid modification. Given the rather low specificity of diethyl pyrocarbonate (DEPC) for histidine modification, we modified Tris-washed PSII membranes with a novel and more specific histidyl modifier, platinum(II) (2,2':6',2"-terpyridine) chloride (Pt-TP). Both the "diphenylcarbazide (DPC)-inhibition assay" and single-turnover flash approaches were used. The concentration dependence of Pt-TP modification on steady-state measurements shows two types of interactions, each accounting for about half of the full effect. At concentrations <50 microM, Pt-TP modifies mostly histidyls and abolishes half of the observed Mn inhibition of DPC-mediated 2,6-dichlorophenolindophenol (DCIP) photoreduction (equivalent to two high-affinity, Mn-binding ligands). This effect can be blocked by addition of Mn2+ during Pt-TP modification. Double-modification experiments with DEPC and Pt-TP demonstrate that both modifiers affect the same observable histidyl residues in PSII. Above 50 microM, Pt-TP modifies mostly cysteines (or histidines in a more hydrophobic environment) and has an additional effect on the reducing side of PSII that (a) does not involve Mn binding and (b) results in the apparent abolishment of all four of the Mn-binding ligands detected by the DPC-inhibition assay. Single-flash experiments show that histidyl modification does not eliminate the binding of the high-affinity, photooxidizable Mn2+ to Asp170 on D1 (nor does it significantly affect high-affinity DPC photooxidation), but it does decrease the binding affinity (Kd) of that Mn from 0.6 to 1.5 microM, particularly at lower (<50 microM Pt-TP) concentrations. Double-modification experiments also demonstrate that the lower affinity, photooxidizable Mn-binding site, uncovered when the high-affinity site is modified with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) [see Ghirardi et al. (1998)], is not associated with a histidyl ligand. Three nonphotooxidizable, high-affinity Mn2+ ions bind to a second carboxyl and two histidyl ligands, and these Mn are not photooxidized by a flash even when the ligand to the photooxidizable Mn is modified by EDC. Proteolytic enzyme studies indicate that the two histidyl ligands identified by the DPC-inhibition assay are probably His337 on D1 and His 339 on D2, but His 332 on D1 is not eliminated.     

Studies of Ca2+ binding in spinach photosystem II using 45Ca2+

The Ca(2+)-binding properties of photosystem II were investigated with radioactive 45Ca2+. PS II membranes, isolated from spinach grown on a medium containing 45Ca2+, contained 1.5 Ca2+ per PS II unit. Approximately half of the incorporated radioactivity was lost after incubation for 30 h in nonradioactive buffer. About 1 Ca2+/PS II bound slowly to Ca(2+)-depleted membranes in the presence of the extrinsic 16- and 23-kDa polypeptides in parallel with restoration of oxygen-evolving activity. The binding was heterogeneous with dissociation constants of 60 microM (0.7 Ca2+/PS II) and 1.7 mM (0.3 Ca2+/PS II), respectively, which could reflect different affinities of the dark-stable S-states for Ca2+. The reactivation of oxygen-evolving activity closely followed the binding of Ca2+, showing that a single exchangeable Ca2+ per PS II is sufficient for the water-splitting reaction to function. In PS II, depleted of the 16- and 23-kDa polypeptides, about 0.7 exchangeable Ca2+/PS II binds with a dissociation constant of 26 microM, while 0.3 Ca2+ binds with a much weaker affinity (Kd > 0.5 mM). The rate of binding of Ca2+ in the absence of the two extrinsic polypeptides was significantly higher than with the polypeptides bound. The rate of dissociation of bound Ca2+ in the dark, which had a half-time of about 80 h in intact PS II, increased in the absence of the 16- and 23-kDa polypeptides and showed a further increase after the additional removal of the 33-kDa protein and manganese. The rate of dissociation was also significantly faster in weak light than in the dark regardless of the presence or absence of the 16- and 23-kDa polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function

Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.     

The interpretation of phlebograms using fibrinogen labeled with 99 mTc

A new method for the detection of deep-vein thrombosis is presented, consisting of a single antecubital injection of fibrinogen labeled with 99mTc. This atraumatic procedure allows one to visualize the large veins of the lower limbs, the venae iliacae and the distal part of the vena cava inferior. This paper discusses how to interpret these phlebograms.     

Investigation of the structure of spinach photosystem II reaction center complex

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.