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.     

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.     

Analysis of the interaction of water with the manganese cluster of photosystem II using isotopically labeled water

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.     

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.     

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.     

The effect of recombinant human growth hormone on regulation of growth hormone secretion and blood glucose in insulin-dependent diabetes

The type 1 copper in Pseudomonas aeruginosa azurin was studied by electron paramagnetic resonance (EPR) spectroscopy at low microwave frequencies. Partially resolved ligand hyperfine structure was observed in the perpendicular region of the spectra at both S-band (2.4 GHz) and L-band (1.1 GHz). A trial and error method, requiring several hundred simulations, has been used to simulate the low frequency EPR data and yield an optimum value of 30 MHz for ACUx, more than one half that previously reported. The fit between the simulated and experimental data is sensitive to changes in the Euler angles and, in particular, to the angle alpha which rotates the Cu A-tensor about the z-axis. Thus, the A- and g-tensors for copper in P. aeruginosa azurin do not appear to be coincident. A value for the Euler angle beta of at least 10 degrees does not disturb the fit between the simulated and experimental data. These studies demonstrate the advantage of evaluating EPR parameters from simulations at more than one frequency, especially at low frequencies where ligand superhyperfine structure may be resolved for type 1 copper.     

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.     

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.     

Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.     

UV-B-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components

Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.     

Light-induced high-spin signals from the oxygen evolving center in Ca2+-depleted photosystem II studied by dual mode electron paramagnetic resonance spectroscopy

Illuminating Ca2+-depleted photosystem (PS) II membranes generated two new EPR signals at g = 11 and 15 by perpendicular and parallel polarization modes, respectively. Two turnovers of the oxygen evolving center (OEC) beyond the modified S2' state are required for the appearance of these signals. The formation of the signals correlated with that of an asymmetric (singlet-like) EPR signal observed at g approximately 2. Spectral simulation indicated that both signals arose from a transition between |2(+/-)> levels with intradoublet splitting of Delta = 0.276 cm-1 in an S = 2 spin system. Furthermore, the two signals in parallel and perpendicular modes were formed at the same time, indicating that the same metal center was responsible. The molecular z-axis of the S = 2 spin system for the signals was almost parallel to the plane of thylakoid membranes. These results indicate that the Mn cluster in the photosynthetic oxygen evolving center is the source of the new EPR species which may be a Mn(IV)-Mn(IV) or Mn(III)-Mn(III) dimer or a Mn(III) monomer. Redox events of the Mn cluster in the Ca2+-depleted PS II are discussed based on these observations.     

NMR and other physical methods

Solid-state NMR can be used for the samples under limited motions such as membrane systems with membrane transport proteins. Nuclear spin interactions such as the chemical shift anisotropy, dipole-dipole interaction and quadrupole interaction can provide the structural information. To get the information at high resolution, the sample should be either oriented or rotated at the magic angle. New methods are developed to recover the nuclear spin interactions under magic angle spinning. By using specifically deuterated phospholipids, detailed information on the protein-lipid interactions can be obtained from the quadrupole splittings of these phospholipids. X-ray crystallography has contributed to the structural analysis of membrane transport proteins after the crystallization method in the presence of detergents was developed.     

Molybdopterin radical in bacterial aldehyde dehydrogenases

The EPR spectra of three different molybdoprotein aldehyde dehydrogenases, one purified from Comamonas testosteroni and two purified from Amycolatopsis methanolica, showed in their oxidized state a novel type of signal. These three enzymes contain two different [2Fe-2S] centers, one flavin and one molybdopterin cytosine dinucleotide, as cofactors all of which are expected to be EPR silent in the oxidized state. The new EPR signal is isotropic with g = 2.004 both at X-band and Q-band frequencies, consists of six partially resolved lines, and shows Curie temperature behavior suggesting that the signal is due to an organic radical with S = 1/2. The EPR spectra of Comamonas testosteroni aldehyde dehydrogenase obtained after cultivation in media containing 15NH4Cl and/or after substitution of H2O for D2O show the presence of both nitrogen and proton hyperfine interactions. Simulations of the spectra of the four possible isotope combinations yield a single set of hyperfine coupling constants. The electron spin shows hyperfine interaction with a single I = 1 (0.9 mT) ascribed to a N nucleus, with a single I = 1/2 (1.5 mT) ascribed to one nonexchangeable H nucleus, and with two, exchangeable, identical I = 1/2 spins (0.6 mT) ascribed to two identical exchangeable protons. Taken together, the observations and simulations rule out amino acid residues or flavin as the origin of the radical. The values of the various hyperfine coupling constants are consistent with the properties expected for a molybdenum(VI)-trihydropterin radical in which the N5 atom is engaged in two hydrogen-bonding interactions with the protein. The majority of the electron (spin) density of the radical is located at and around the N5 atom and at the proton bound to the C6 atom of the pterin ring. The EPR spectrum of the molybdopterin radical broadens above 65 K and is no longer detectable above 168 K, indicating that it is not magnetically isolated. The line broadening is ascribed to cross-relaxation with a nearby, rapidly relaxing, oxidized [2Fe-2S] center involving its magnetic S = 1 excited state in this process. The amount of radical was apparently not changed by addition of aldehydes or oxidants, but it disappeared upon reduction by sodium dithionite. Therefore, whether the molybdenum(VI) trihydropterin radical as detected here is a functional intermediate in catalysis remains to be investigated further.     

Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy

Thromboxane A2 (TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 micromol of TXA2/min/mg of protein at 23 degreesC. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (Kd approximately 0.5 microM), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.     

Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2. Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.     

NO reversibly reduces the water-oxidizing complex of photosystem II through S0 and S-1 to the state characterized by the Mn(II)-Mn(III) multiline EPR signal

Incubation of photosystem II preparations with NO at -30 degreesC results in the slow formation of a unique state of the water-oxidizing complex (WOC), which was recently identified as a Mn(II)-Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. Evolution of the Mn(II)-Mn(III) EPR signal proceeds through one or more intermediates [Goussias, C., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261-9266]. In an effort to identify these intermediates, we have examined the time course of the signal evolution in the presence and absence of methanol. An early step of the interaction of NO with the WOC is the reduction of S1 to the S0 state, characterized by the weak Mn-hyperfine structure recently reported for that state. At longer times S0 is further reduced to a state which has the properties of the S-1 state, in that single-turnover illumination restores the S0 signal. The Mn(II)-Mn(III) state forms after the S-1 state and is tentatively assigned to an (iso)S-2 state, although lower states or a modified S-1 state cannot be excluded at present. Following removal of NO 60-65% of the initial S2 multiline signal size or the O2-evolving activity can be restored. The data provide for the first time EPR information on a state lower than S0. Furthermore, the low-temperature NO treatment provides a simple means for the selective population of the S0, S-1 and the Mn(II)-Mn(III) states.     

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.     

Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase

Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands. In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pKa for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.     

A glutamate bridge is essential for dimer stability and metal selectivity in manganese superoxide dismutase

In Escherichia coli manganese superoxide dismutase (MnSOD), the absolutely conserved Glu170 of one monomer is hydrogen-bonded to the Mn ligand His171 of the other monomer, forming a double bridge at the dimer interface. Point mutation of Glu170 --> Ala destabilizes the dimer structure, and the mutant protein occurs as a mixture of dimer and monomer species. The purified E170A MnSOD contains exclusively Fe and is devoid of superoxide dismutase activity. E170A Fe2-MnSOD closely resembles authentic FeSOD in terms of spectroscopic properties, anion interactions and pH titration behavior. Reconstitution of E170A Fe2-MnSOD with Mn(II) salts does not restore superoxide dismutase activity despite the spectroscopic similarity between E170A Mn2-MnSOD and wild type Mn2-MnSOD. Growth of sodA+ and sodA- E. coli containing the mutant plasmid pDT1-5(E170A) is impaired, suggesting that expression of mutant protein is toxic to the host cells.     

Fosfomycin resistance protein (FosA) is a manganese metalloglutathione transferase related to glyoxalase I and the extradiol dioxygenases

The enzyme conferring resistance to the antibiotic fosfomycin [(1R,2S)-1,2-epoxypropylphosphonic acid] originally reported by Suarez and co-workers [Area, P., Hardisson, C., & Suarez, J. E. (1990) Antimicrob. Agents Chemother. 34, 844-848] is demonstrated in this study to be a metalloglutathione transferase. The apoenzyme is a dimer of 16 kDa subunits. Electron paramagnetic resonance spectroscopy and water proton nuclear magnetic resonance longitudinal relaxation rates suggest that each subunit contains a mononuclear Mn2+ center that interacts strongly with the substrate fosfomycin (Kd = 17 microM) more weakly with the product (Kd = 1.1 mM) and very weakly or not at all with GSH. Inhomogeneous broadening of the EPR signals of enzyme-bound Mn2+ in the presence of H2(17)O indicates that three of the coordination sites on the metal are occupied by water. Sequence alignments, three-dimensional structures, and mechanistic considerations suggest that FosA is related to at least two other metalloenzymes, glyoxalase I and the Mn2+- or Fe2+-containing extradiol dioxygenases. The mechanistic imperative driving the evolution of this previously unidentified superfamily of metalloenzymes is proposed to be bidentate coordination of a substrate or intermediate to the metal center in the enzyme-catalyzed reactions.