Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: II. A binuclear manganese-containing 34 kilodalton protein,a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O₂-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O₂ evolution activity from functional membranes, such as incubation in 1.5 mM NH₂OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two ⁵⁵Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn₂(III,III) site or an antiferromagnetically coupled mixed-valence Mn₂(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH₂OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn₂(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn₂(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O₂ evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O₂-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O₂ evolving complex.     

The first spectroscopic model for the S1 state multiline signal of the OEC

The parallel-mode electron paramagnetic resonance (EPR) spectrum of the S(1) state of the oxygen-evolving complex (OEC) shows a multiline signal centered around g=12, indicating an integer spin system. The series of [Mn(2)(2-OHsalpn)(2)] complexes were structurally characterized in four oxidation levels (Mn(II)(2), Mn(II)Mn(III), Mn(III)(2), and Mn(III)Mn(IV)). By using bulk electrolysis, the [Mn(III)Mn(IV)(2-OHsalpn)(2)(OH)] is oxidized to a species that contains Mn(IV) oxidation state as detected by X-ray absorption near edge spectroscopy (XANES) and that can be formulated as Mn(IV)(4) tetramer. The parallel-mode EPR spectrum of this multinuclear Mn(IV)(4) complex shows 18 well-resolved hyperfine lines center around g=11 with an average hyperfine splitting of 36 G. This EPR spectrum is very similar to that found in the S(1) state of the OEC. This is the first synthetic manganese model complex that shows an S(1)-like multiline spectrum in parallel-mode EPR.     

Consistent simulation of X- and Q-band EPR spectra of an unsymmetric dinuclear Mn2(II,III) complex

Simulation of X- and Q-band electron paramagnetic resonance (EPR) spectra of an unsymmetric dinuclear [Mn(2)(II,III)L(mu-OAc)(2)]ClO(4) complex (1), (L is the dianion of 2-{[N,N-bis(2-pyridylmethyl)amino]methyl}-6-{[N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N-(2-pyridylmethyl)amino]methyl}-4-methylphenol) was performed using one consistent set of simulation parameters. Rhombic g-tensors and hyperfine tensors were necessary to obtain satisfactory simulation of the EPR spectra. The anisotropy of the effective hyperfine tensors of each individual (55)Mn ion was further analyzed in terms of intrinsic hyperfine tensors. Detailed analysis shows that the hyperfine anisotropy of the Mn(III) ion is a result of the Jahn-Teller effect and thus an inherent character. In contrast, the anomalous hyperfine anisotropy of the Mn(II) ion is attributed as being transferred from the Mn(III) ion through the spin exchange interaction. The anisotropy parameter for the Mn(II) is deduced as D(II)=-1.26+/-0.2cm(-1). This is the first reported D(II) value for a Mn(II) ion in a weakly exchange coupled mixed-valence Mn(2)(II,III) complex with a bis-mu-acetato-bridge. The [see text] electronic configuration of the Mn(III) ion in 1 is revealed by the negative sign of its intrinsic hyperfine tensor anisotropy, Deltaa(III)=a(z)-a(x,y)=-46cm(-1). Lower spectral resolution of the Q-band EPR spectrum as compared to the X-band EPR spectrum is associated to large line width broadening of the x- and y-components in contrast to the z-component. The origins of the unequal distribution of line width between the z- and x-, y-components are discussed.     

A mixed valence form of the iron cluster in the B2 protein of ribonucleotide reductase from Escherichia coli.

A mixed valent form of the iron cluster (Fe(II)Fe(III) in the B2 protein of ribonucleotide reductase has been isolated and characterized. The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum. This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleotide reductase.     

A calcium-specific site influences the structure and activity of the manganese cluster responsible for photosynthetic water oxidation

EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes--implications for photosynthetic water-oxidation

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem II (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N(3)O(3) in 1, N(2)O(4) in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(II)<-->Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(III). In 2: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)<-->Mn(III)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(IV). In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at approximately 1V, but the mu-O(H) bridge is observed at the Mn(2)(III,III) level in 1 and at the Mn(III,IV) level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential.     

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.     

Orientation of the Mn(II)-Mn(III) dimer which results from the reduction of the oxygen-evolving complex of photosystem II by NO: an electron paramagnetic resonance study

The central part of the oxygen-evolving complex of photosystem II is a cluster of four manganese atoms. The known EPR spectra in the various oxidation states of the cluster are complicated by the magnetic interactions of the four Mn ions and accordingly are difficult to analyze. It has been shown recently that NO at -30 degrees C slowly reduces the cluster to a Mn(II)-Mn(III) state [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587). We study herein the orientation dependence of the Mn(II)-Mn(III) EPR spectrum with respect to the thylakoid membrane plane. Both the powder and the oriented spectra are satisfactorily simulated with the same set of fine and hyperfine parameters assuming axial symmetry and collinear g and A tensors. The axial component of the tensors is found to be oriented at an angle of 20 degrees +/- 10 degrees to the membrane plane normal (mosaic spread Omega = 40 degrees ). We make the reasonable assumption that the Mn(II)-Mn(III) dimer is one of the di-mu-oxo units that has been suggested to comprise the Mn tetramer. On the basis of the sign of the hyperfine tensor anisotropy, the axial direction is assigned to the d(z(2)) orbital of Mn(III), which by comparison with synthetic model complexes is assumed to be oriented perpendicular to the Mn-(mu-oxo)-Mn plane. The present results complement earlier orientation studies by EXAFS and suggest that the Mn-(mu-oxo)-Mn plane makes a small angle (approximately 20 degrees) with the membrane plane and the axis connecting the bridging oxygens is approximately parallel to the plane.     

Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.     

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.     

Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center

Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyces graminis, and the oxygenation mechanism was investigated. A kinetic isotope effect, k(H)/k(D) = 21-24, was observed with [U-(2)H]linoleic acid as a substrate. The relative biosynthesis of (11S)-hydroperoxylinoleate (11S-HPODE) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U-(2)H]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha-linolenic acids indicated catalytic lag times of approximately 45 ms, which were followed by bursts of enzyme activity for approximately 60 ms and then by steady state (k(cat) approximately 26 and approximately 47 s(-1), respectively). 11S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed from linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accompanied by collisional quenching of the long wavelength fluorescence (640-685 nm) by fatty acid substrates and 13R-HPODE. Electron paramagnetic resonance (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine splitting superimposed on a broad resonance indicating two populations of Mn(II) bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated approximately 0.8 Mn(2+) atoms/lipoxygenase molecule. These observations are consistent with a mononuclear Mn(II) center in the native state, which is converted during catalysis to an EPR silent Mn(III) state. We propose that manganese lipoxygenase has kinetic and redox properties similar to iron lipoxygenases.     

High-field (94-GHz) EPR spectroscopy on the S(2) multiline signal of Photosystem II

The multiline signal of the S(2) state in Photosystem II was measured both in frozen-solution and single-crystal preparations from the cyanobacterium Thermosynechococcus elongatus. The frozen-solution EPR spectrum shows a gaussian-like line shape without any resolution of Mn hyperfine couplings. This line shape can be understood on the basis of the single-crystal spectra, where a strong orientation dependence of partially resolved hyperfine structures appears. Simulation of the frozen-solution spectrum on the basis of Mn hyperfine couplings taken from published pulse-ENDOR data yields a fully rhombic g-matrix for the multiline signal with principal components 1.997, 1.970, and 1.965. The resulting isotropic g-value g(iso)=1.977 is surprisingly small compared to other manganese complexes containing manganese ions in the formal oxidation states three and four.     

Presence in Photosystem II Core Complexes of a 34-Kilodalton Polypeptide Required for Water Photolysis

Photosystem II (PSII) reaction center core complexes have been isolated and characterized from wild type (WT) Scenedesmus obliquus and from its LF-1 mutant. LF-1 thylakoids are blocked on the oxidizing side of PSII and have a reduced Mn content. Visible absorption and low temperature fluorescence spectra of both core complexes are identical and resemble those reported for spinach (Satoh, Butler 1978 Plant Physiol 61: 373-379). Lithium dodecyl sulfate-polycrylamide gel electrophoresis reveals that a protein alteration, originally observed in thylakoid membranes (Metz, Wong, Bishop 1980 FEBS Lett 114: 61-66), is retained in the PSII core particles. That is, a 34-kilodalton (kD) polypeptide, present in the WT core complex, is missing in the mutant, and the core complex of the mutant contains a 36-kD protein not present in the WT. The 34-kD intrinsic protein is also observed in O₂-evolving PSII preparations and PSII core complexes from spinach. It is distinct from the 33-kD extrinsic protein first reported by T. Kuwabara and N. Murata (1979 Biochim Biophys Acta 581: 228-236). We suggest that the 34-kD protein is a site of Mn binding in the PSII membrane.     

Non-heme manganese catalase--the 'other' catalase

Non-heme manganese catalases are widely distributed over microbial life and represent an environmentally important alternative to heme-containing catalases in antioxidant defense. Manganese catalases contain a binuclear manganese complex as their catalytic active site rather than a heme, and cycle between Mn(2)(II,II) and Mn(2)(III,III) states during turnover. X-ray crystallography has revealed the key structural elements of the binuclear manganese active site complex that can serve as the starting point for computational studies on the protein. Four manganese catalase enzymes have been isolated and characterized, and the enzyme appears to have a broad phylogenetic distribution including both bacteria and archae. More than 100 manganese catalase genes have been annotated in genomic databases, although the assignment of many of these putative manganese catalases needs to be experimentally verified. Iron limitation, exposure to low levels of peroxide stress, thermostability and cyanide resistance may provide the biological and environmental context for the occurrence of manganese catalases.     

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.     

Evidence for an association between a 33 kDa extrinsic membrane protein,manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S₁ → S₂ reaction, as seen by the multiline EPR signal for the S₂ state of the water-oxidizing complex, while reversibly inhibiting O₂ evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O₂ evolution and the S₂ multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl₂ to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O₂ evolution and S₂ multiline yield. These depleted membranes do not oxidize water or produce the normal S₂ state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.     

Inactivation and reactivation of manganese catalase: oxidation-state assignments using X-ray absorption spectroscopy.

The oxidation states of the Mn atoms in three derivatives of Mn catalase have been characterized using a combination of X-ray absorption near-edge structure (XANES) and EPR spectroscopies. The as-isolated enzyme has an average oxidation state of Mn(III) and contains a Mn(III) form, together with a reduced Mn(II) form and a variable amount (10-25%) of a Mn(III)/Mn(IV) mixed-valence derivative. Treatment with NH2OH rapidly reduces the majority of the enzyme to a Mn(II) derivative with no loss of activity. Inactivation by treatment with NH2OH + H2O2 converts all of the enzyme to a mixed-valence Mn(III)/Mn(IV) form. The inactive, mixed-valence derivative can be completely reactivated by long-term (greater than 1 h) anaerobic incubation with NH2OH, giving a reduced Mn(II)/Mn(II) derivative. These data suggest a catalytic model in which the enzyme cycles between a reduced Mn(II)/Mn(II) state and an oxidized Mn(III)/Mn(III) state.     

Photo-induced oxidation of a dinuclear Mn(2)(II,II) complex to the Mn(2)(III,IV) state by inter- and intramolecular electron transfer to Ru(III)tris-bipyridine

To model the structural and functional parts of the water oxidizing complex in Photosystem II, a dimeric manganese(II,II) complex (1) was linked to a ruthenium(II)tris-bipyridine (Ru(II)(bpy)(3)) complex via a substituted L-tyrosine, to form the trinuclear complex 2 [J. Inorg. Biochem. 78 (2000) 15]. Flash photolysis of 1 and Ru(II)(bpy)(3) in aqueous solution, in the presence of an electron acceptor, resulted in the stepwise extraction of three electrons by Ru(III)(bpy)(3) from the Mn(2)(II,II) dimer, which then attained the Mn(2)(III,IV) oxidation state. In a similar experiment with compound 2, the dinuclear Mn complex reduced the photo-oxidized Ru moiety via intramolecular electron transfer on each photochemical event. From EPR it was seen that 2 also reached the Mn(2)(III,IV) state. Our data indicate that oxidation from the Mn(2)(II,II) state proceeds stepwise via intermediate formation of Mn(2)(II,III) and Mn(2)(III,III). In the presence of water, cyclic voltammetry showed an additional anodic peak beyond Mn(2)(II,III/III,III) oxidation which was significantly lower than in neat acetonitrile. Assuming that this peak is due to oxidation to Mn(2)(III,IV), this suggests that water is essential for the formation of the Mn(2)(III,IV) oxidation state. Compound 2 is a structural mimic of the water oxidizing complex, in that it links a Mn complex via a tyrosine to a highly oxidizing photosensitizer. Complex 2 also mimics mechanistic aspects of Photosystem II, in that the electron transfer to the photosensitizer is fast and results in several electron extractions from the Mn moiety.     

Comparative effects of tumour necrosis factor-alpha (cachectin), interleukin-1-beta and tumour growth on amino acid metabolism in the rat in vivo. Absorption and tissue uptake of alpha-amino[1-14C]isobutyrate.

Incubation of a membrane preparation enriched in Photosystem Two (PSII) at alkaline pH inhibited the water-splitting reactions in two distinct steps. Up to pH 8.5 the inhibition was reversible, whereas at higher alkalinities it was irreversible. It was shown that the reversible phase correlated with loss and rebinding of the 23 kDa extrinsic polypeptide. However, after mild alkaline treatments a partial recovery was possible without the binding of the 23 kDa polypeptide when the assay was at the optimal pH of 6.5 and in a medium containing excess Cl-. The irreversible phase was found to be closely linked with the removal of the 33 kDa extrinsic protein of PSII. Treatments with pH values above 8.5 not only caused the 33 kDa protein to be displaced from the PSII-enriched membranes, but also resulted in an irreversible modification of the binding sites such that the extrinsic 33 kDa protein could not reassociate with PSII when the pH was lowered to 6.5. The results obtained with these more extreme alkaline pH treatments support the notion that the 23 kDa protein cannot bind to PSII unless the 33 kDa protein is already bound. The differential effect of pH on the removal of the 23 kDa and 33 kDa proteins contrasted with the data of Kuwabara & Murata [(1983) Plant Cell Physiol. 24, 741-747], but this discrepancy was accounted for by the use of glycerol in the incubation media.