Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002.

For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that N-acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gadA gene from Synechocystis sp. PCC 6803 was heterologously expressed in Synechococcus sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven Synechococcus sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in Synechococcus sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.

Bicarbonate is a native cofactor for assembly of the manganese cluster of the photosynthetic water oxidizing complex. Kinetics of reconstitution of O2 evolution by photoactivation.

Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) of the water oxidizing enzyme of oxygenic photosynthesis generates O(2) evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn(2+) concentrations. The first step, the binding and photooxidation of Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition). Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.

A functional role for tyrosine-D in assembly of the inorganic core of the water oxidase complex of photosystem II and the kinetics of water oxidation.

The role of D2-Tyr160 (Y(D)), a photooxidizable residue in the D2 reaction center polypeptide of photosystem II (PSII), was investigated in both wild type and a mutant strain (D2-Tyr160Phe) in which phenylalanine replaces Y(D) in the cyanobacterium Synechocystis sp. (strain PCC 6803). Y(D) is the symmetry-related tyrosine that is homologous to the essential photoactive Tyr161(Y(Z)) of the D1 polypeptide of PSII. We compared the flash-induced yield of O(2) in intact, functional PSII centers from both wild-type and mutant PSII core complexes. The yield of O(2) in the intact holo-enzyme was found to be identical in the mutant and wild-type PSII cores using long (saturating) pulses or continuous illumination, but was observed to be appreciably reduced in the mutant using short (nonsaturating) light pulses (<50 ms). We also compared the rates of the first two kinetically resolved steps of photoactivation. Photoactivation is the assembly process for binding of the inorganic cofactors to the apo-water oxidation/PSII complex (apo-WOC-PSII) and their light-induced photooxidation to form the functional Mn(4)Ca(1)Cl(x)() core required for O(2) evolution. We show that the D2-Tyr160Phe mutant cores can assemble a functional WOC from the free inorganic cofactors, but at a much slower rate and with reduced quantum efficiency vs wild-type PSII cores. Both of these observations imply that the presence of Y(D)(*) leads to a more efficient photooxidation of the Mn cluster relative to deactivation (reductive processes). One possible explanation for this behavior is that the phenolic proton on Y(D) is retained within the reaction center following Y(D) oxidation. The positive charge, likely shared by D2-His189 and other residues, raises the reduction potential of P(680)(+)/P(680), thereby increasing the driving force for the oxidation of Mn(4)Y(Z). There is, therefore, a competitive advantage to organisms that retain the Y(D) residue, possibly explaining its retention in all sequences of psbD (encoding the D2 polypeptide) known to date. We also find that the sequence of metal binding steps during assembly of apo-WOC-PSII centers in cyanobacteria cores differs from that in higher plants. This is seen by a reduced calcium affinity at its effector site and reduced competition for binding to the Mn(II) site, resulting in acceleration of the initial lagtime by Ca(2+), in contrast to retardation in spinach. Ca(2+) binding to its effector site promotes the stability of the photointermediates (IM1 and above) by suppressing unproductive decay.

The origin of atmospheric oxygen on Earth: the innovation of oxygenic photosynthesis.

The evolution of O(2)-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO(2)) is a more efficient alternative substrate than water for O(2) production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O(2) production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O(2) evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.

The inorganic biochemistry of photosynthetic oxygen evolution/water oxidation.

At the request of the organizer of this special edition, we have attempted to do several things in this manuscript: (1) we present a mini-review of recent, selected, works on the light-induced inorganic biogenesis (photoactivation), composition and structure of the inorganic core responsible for photosynthetic water oxidation; (2) we summarize a new proposal for the evolutionary origin of the water oxidation catalyst which postulates a key role for bicarbonate in formation of the inorganic core; (3) we summarize published studies and present new results on what has been learned from studies of 'inorganic mutants' in which the endogenous cofactors (Mn(n+), Ca2+, Cl-) are substituted; (4) the first DeltapH changes measured during the photoactivation process are reported and used to develop a model for the stepwise photo-assembly process; (5) a comparative analysis is given of data in the literature on the kinetics of substrate water exchange and peroxide binding/dismutation which support a mechanistic model for water oxidation in general; (6) we discuss alternative interpretations of data in the literature with a view to forecast new avenues where progress is needed.

Bicarbonate accelerates assembly of the inorganic core of the water-oxidizing complex in manganese-depleted photosystem II: a proposed biogeochemical role for atmospheric carbon dioxide in oxygenic photosynthesis.

The proposed role for bicarbonate (HCO(3)(-)) as an intrinsic cofactor within the water-oxidizing complex (WOC) of photosystem II (PSII) [Klimov et al. (1997) Biochemistry 36, 16277-16281] was tested by investigation of its influence on the kinetics and yield of photoactivation, the light-induced assembly of the functional inorganic core (Mn(4)O(y)Ca(1)Cl(x)) starting from the cofactor-depleted apo-WOC-PSII center and free Mn(2+), Ca(2+), and Cl(-). Two binding sites for bicarbonate were found that stimulate photoactivation by accelerating the formation and suppressing the decay, respectively, of the first light-induced assembly intermediate, IM(1) [apo-WOC-Mn(OH)(2)(+)]. A high-affinity bicarbonate site (K(D)

"Bridging hydroxide effect" on mu-carboxylato coordination and electrochemical potentials of bimetallic centers: Mn2(II,II) and Mn2(III,III) complexes as functional models of dimanganese catalases.

Synthesis, solution structures, and electrochemistry of several dinuclear Mn2(II,II) complexes (1-4) and Mn2(III,III) complexes (6 and 8), derived from a functional catalase mimic [(L1,2)Mn2(II,II)(mu 13-O2CCH3)]2+ (1) are described that enable testing of the role of intramolecular hydroxide ligands on the redox properties. Addition of 1 equiv of hydroxide to 1 or 3 forms [(L1,2)Mn2(II,II)(mu 13-O2CCH3)(mu- OH)]+ (7A or 7B, respectively), possessing two six-coordinate Mn(II) ions bridged by hydroxide and acetato ligands. Two-electron oxidation of 7 with O2 occurs by forming [(L1,2)Mn2(III,III)(mu 1,3-O2CCH3)(mu-OH)]3+ (8) and H2O2 with no ligand rearrangements in methanol. Reaction of 8 with 2-3 equiv hydroxide forms [(L1,2)Mn2(III,III)(mu-O)(OH)(O2CCH3)]+ in which deprotonation of mu-OH- to yield mu-O2- favors subsequent addition of a terminal hydroxide ligand, accommodated by a bridging-to-terminal "carboxylate-shift". Preservation of six-coordinate Mn(II) ions throughout all hydroxide-induced transformations is observed, including oxidation by O2. Cyclic voltammetry reveals that addition of mu-OH- converts the two-electron redox couple II,II/III,III for complexes 1-4 to sequential one-electron couples at lower reduction potentials, yielding substantial stabilization of the II,III and III,III oxidation states by delta E = 440 and 730 mV, respectively. Binding of a second OH- to 7A or 7B forms (L1,2)Mn2(II,II)(mu 13-O2CCH3)(OH)2, containing two six-coordinate Mn(II) ions with two terminal hydroxides and a mu 1,3-bridging acetato. Electrochemistry reveals that displacement of the bridging hydroxide to a terminal site upon addition of the second OH- restores a two-electron redox couple II,II/III,III but now at a higher reduction potential with considerable loss of the electrochemical stabilization energy provided by the mu-OH- (delta E = 250 and 350 mV loss for Mn2(II,II) and Mn2(III,III), (respectively). These results indicate a considerably stronger influence of bridging vs terminal hydroxide ligands in stabilizing the higher oxidation states and separating the one-electron redox potentials of bimetallic centers. By contrast, in the absence of mu-OH- bridges the longer separation with the mu 1,3-carboxylato bridge in dimanganese(II,II) complexes leads to nearly complete uncoupling of the Mn(II) oxidation potentials, thus yielding a two-electron redox transition to (III,III). We hypothesize that this "bridging hydroxide effect" may be due to both greater screening of the repulsive intermetallic electric potential energy and increased resonance stabilization of the mixed-valence (II,III) oxidation state by charge delocalization . These data provide a physicochemical basis for interpretation of the catalase activity of these complexes and of dimanganese catalase enzymes (see the following manuscript).

Mechanism of hydrogen peroxide dismutation by a dimanganese catalase mimic: dominant role of an intramolecular base on substrate binding affinity and rate acceleration.

Several modifications of the manganese coordination environment and oxidation states of a family of synthetic dimanganese complexes have been introduced in search of the structural features that promote high rates of hydrogen peroxide dismutation (catalase activity). The X-ray structure of reduced catalase (T thermophilus) reveals a dimanganese(II,II) site linked by three bridges: mu 13-glutamate-, mu-OH-, and mu-OH2. The roles of a bridging hydroxide vs mu-aqua and the carboxylate have been examined in the reduced Mn2(II,II) complexes, [(L1,2)Mn2(mu-O2CCH3)(mu-X)]2+ for X- = OH- (7A) or X = H2O (1-4), and their oxidized Mn2(III,III) analogues, [(L1,2)Mn2(mu-O)(O2CCH3)(OH)]+ (6) (L1 is N,N,N',N'-tetrakis(2-methylenebenzamidazolyl)-1,3-diaminopropan- 2-ol, and L2 is the tetrakis-N-ethylated analogue of L1, which has all amine protons replaced by ethyl groups). The steady-state catalase rate is first-order in concentration of both substrate and reduced catalyst and saturates at high peroxide concentrations in all cases, confirming peroxide/catalyst complex formation. No catalyst decomposition is seen after > 2000 turnovers. Catalysis proceeds via a ping-pong mechanism between the Mn2(II,II/III,III) redox states, involving complexes 6 and 7A/7A'. The Mn2(III,IV) oxidation state was not active in catalase activity. Replacement of the mu-aqua bridge by mu-hydroxide eliminates a kinetic lag phase in production of the O2 product, increases the affinity for substrate peroxide in the rate-limiting step as seen by a 5-fold. decrease in the Michaelis constant (KM), and accelerates the maximum rate (kcat) by 65-fold The kinetic and spectroscopic data are consistent with substrate deprotonation by the hydroxide bridge, yielding a hydroperoxyl bridge coordinated between the Mn ions (mu, eta 2 geometry, "end-on") as the basis for catalysis: mu-OH- + H2O2-->mu-O2H- + H2O. Binding of a second hydroxide ion to 7A causes a further increase in kcat by 4-fold with no further change in substrate affinity (KM). By contrast, free (noncoordinating) bases in solution have no effect on catalysis, thus establishing intramolecular sites for both functional hydroxide anions. Solution structural studies indicate that the presence of 2-5 equiv of hydroxide in solution leads to formation of a bishydroxide species, [(L1,2)Mn2(mu 13-O2CCH3)(OH)2], which in the presence of air or oxygen auto-oxidizes to yield complex 6, a Mn2(III,III)(mu-O) species. Complex 6 oxidizes H2O2 to O2 without a kinetic lag phase and is implicated as the active form of the oxidized catalyst. A maximum increase by 240-fold in catalytic efficiency (kcat/KM = 700 s-1 M-1) is observed with the bishydroxide species versus the aquo complex 1, or only 800-fold less efficient than the enzyme. Deprotonation of the amine groups of the chelate ligand L was shown not to be involved in the hydroxide effects because identical results were obtained using the catalyst with tetrakis(N-ethylated)-L. Uncoupling of the Mn(II) spins by protonation of the alkoxyl bridge (LH) was observed to lower the catalase activity. Comparisons to other dimanganese complexes reveals that the Mn2(II,II)/Mn2(III,III) redox potential is not the determining factor in the catalase rate of these complexes. Rather, rate acceleration correlates with the availability of an intramolecular hydroxide for substrate deprotonation and with binding of the substrate at the bridging site between Mn ions in the reductive O-O bond cleavage step that forms water and complex 6.

Conversion of core oxos to water molecules by 4e-/4H+ reductive dehydration of the Mn4O2(6+) core in the manganese-oxo cubane complex Mn4O4(Ph2PO2)6: a partial model for photosynthetic water binding and activation.

Reaction of the Mn4O4(6+) "cubane" core complex, Mn4O4L6 (1) (L = diphenylphosphinate, Ph2PO2-), with a hydrogen atom donor, phenothiazine (pzH), forms the dehydrated cluster Mn4O2L6 (2), which has lost two mu-oxo bridges by reduction to water (H2O). The formation of 2 was established by electrospray mass spectrometry, whereas FTIR spectroscopy confirmed the release of water molecules into solution during the reduction of 1. UV-vis and EPR spectroscopies established the stoichiometry and chemical form of the pzH product by showing the production of 4 equiv of the neutral pz radical. By contrast, the irreversible decomposition of 1 to individual Mn(II) ions occurs if the reduction is performed using electrons provided by various proton-lacking reductants, such as cobaltocene or electrochemical reduction. Thus, cubane 1 undergoes coupled four-electron/four-proton reduction with the release of two water molecules, a reaction formally analogous to the reverse sequence of the steps that occur during photosynthetic water oxidation leading to O2 evolution. 1H NMR of solutions of 2 reveal that all six of the phosphinate ligands exhibit paramagnetic broadening, due to coordination to Mn ions, and are magnetically equivalent. A symmetrical core structure is thus indicated. We hypothesize that this structure is produced by the dynamic averaging of phosphinato ligand coordination or exchange of mu-oxos between vacant mu-oxo sites. The paramagnetic 1H NMR of water molecules in solution shows that they are able to freely exchange with water molecules that are bound to the Mn ion(s) in 2, and this exchange can be inhibited by the addition of coordinating anions, such as chloride. Thus, 2 possesses open or labile coordination sites for water and anions, in contrast to solutions of 1, which reveal no evidence for water coordination. Complex 2 exhibits greater paramagnetism than that of 1, as seen by 1H NMR, and it possesses a broad (440 G wide) EPR absorption, centered at g = 2, that follows a Curie-Weiss temperature dependence (10-40 K) and is visible only at low temperatures, compared to EPR-silent 1. Its comparison to a spin-integration standard reveals that 2 contains 2 equiv of Mn(II), which is in agreement with the formal oxidation state of 2Mn(II)2Mn(III) that was derived from the titration. The EPR and NMR data for 2 are consistent with a loss of two of the intermanganese spin-exchange coupling pathways, versus 1, which results in two "wingtip" Mn(II) S = 5/2 spins that are essentially magnetically uncoupled from the diamagnetic Mn2O2 base. Bond-enthalpy data, which show that O2 evolution via the reaction 1-->2 + O2, is strongly favored thermodynamically but is not observed in the ground state due to an activation barrier, are included. This activation barrier is hypothesized to arise, in part, from the constraining effect of the facially bridging phosphinate ligands.

Remarkable affinity and selectivity for Cs+ and uranyl (UO22+) binding to the manganese site of the apo-water oxidation complex of photosystem II.

The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP). Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding of large alkali cations (Cs+ >> Rb+ > K+ > Na+ > Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (>/=5000) for any known chelator. Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ >> Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me2+aq --> [MeOH]+aq + H+aq. Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH]+ is the active species that forms upon binding of Mn2+aq to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH]+, a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)2Mn]3+ or apo-WOC-[Mn(mu-OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)2]+). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay prior to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH]+.

L-arginine binding to liver arginase requires proton transfer to gateway residue His141 and coordination of the guanidinium group to the dimanganese(II,II) center.

Rat liver arginase contains a dimanganese(II,II) center per subunit that is required for catalytic hydrolysis of l-arginine to form urea and l-ornithine. A recent crystallographic study has shown that the Mn2 center consists of two coordinatively inequivalent manganese(II) ions, MnA and MnB, bridged by a water (hydroxide) molecule and two aspartate residues [Kanyo et al. (1996) Nature 383, 554-557]. A conserved residue, His141, is located near the proposed substrate binding region at 4.2 A from the bridging solvent molecule. The present EPR studies reveal that there is no essential alteration of the Mn2 site upon mutation of His141 to an Asn residue, which lacks a potential acid/base residue, while the catalytic activity of the mutant enzyme is 10 times lower vs wild-type enzyme. The binding affinity of l-lysine, l-arginine (substrate), and Nomega-OH-l-arginine (type 2 binders) increases inversely with the pKa of the side chain. Binding of l-lysine is more than 10 times weaker, and the substrate Michaelis constant (Km) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and Nomega-OH-L-arginine, type 2 binders, induce extensive loss of the EPR intensity, suggesting direct coordination to the Mn2 center. From these data and the pH dependence of type 2 binders, we conclude that His141 functions as the base for deprotonation of the side-chain amino group of L-lysine and the substrate guanidinium group, -NH-C(NH2)2+ and that the unprotonated side chain of these amino acids is responsible for binding to the active site. A different class of inhibitors (type 1), including L-isoleucine, L-ornithine, and L-citrulline, suppresses enzymatic activity, producing only minor change in the zero-field splitting of the Mn2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 alpha-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the alpha-aminocarboxylate groups of the substrate. A new mechanism for the arginase-catalyzed hydrolysis of L-arginine is proposed which has general relevance to all binuclear hydrolases: (1) Deprotonation of substrate l-arginine(H+) by His141 permits entry of the neutral guanidinium group into the buried Mn2 region. Binding of the substrate imino group (>C=NH), most likely to MnB, is coupled to breaking of the MnB-(mu-H2O) bond, forming a terminal aquo ligand on MnA. (2) Proton transfer from the terminal MnA-aqua ligand to the substrate Ndelta-guanidino atom forms the nucleophilic hydroxide on MnA and the cationic NdeltaH2+-guanidino leaving group. Protonation of the substrate -NdeltaH2+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the MnA-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the Cepsilon-NdeltaH2+ bond, yielding urea and L-ornithine(H+).

Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer.

Formate dehydrogenase H, FDH(Se), from Escherichia coli contains a molybdopterin guanine dinucleotide cofactor and a selenocysteine residue in the polypeptide. Oxidation of 13C-labeled formate in 18O-enriched water catalyzed by FDH(Se) produces 13CO2 gas that contains no 18O-label, establishing that the enzyme is not a member of the large class of Mo-pterin-containing oxotransferases which incorporate oxygen from water into product. An unusual Mo center of the active site is coordinated in the reduced Mo(IV) state in a square pyramidal geometry to the four equatorial dithiolene sulfur atoms from a pair of pterin cofactors and a Se atom of the selenocysteine-140 residue [Boyington, J. C., Gladyshev, V. N., Khangulov, S. V., Stadtman, T. C., and Sun, P. D. (1997) Science 275, 1305-1308]. EPR spectroscopy of the Mo(V) state indicates a square pyramidal geometry analogous to that of the Mo(IV) center. The strongest ligand field component is likely the single axial Se atom producing a ground orbital configuration Mo(dxy). The Mo-Se bond was estimated to be covalent to the extent of 17-27% of the unpaired electron spin density residing in the valence 4s and 4p selenium orbitals, based on comparison of the scalar and dipolar hyperfine components to atomic 77Se. Two electron oxidation of formate by the Mo(VI) state converts Mo to the reduced Mo(IV) state with the formate proton, Hf+, transferring to a nearby base Y-. Transfer of one electron to the Fe4S4 center converts Mo(IV) to the EPR detectable Mo(V) state. The Y- is located within magnetic contact to the [Mo-Se] center, as shown by its strong dipolar 1Hf hyperfine couplings. Photolysis of the formate-induced Mo(V) state abolishes the 1Hf hyperfine splitting from YHf, suggesting photoisomerizaton of this group or phototransfer of the proton to a more distant proton acceptor group A-. The minor effect of photolysis on the 77Se-hyperfine interaction with [77Se] selenocysteine suggests that the Y- group is not the Se atom, but instead might be the imidazole ring of the His141 residue which is located in the putative substrate-binding pocket close to the [Mo-Se] center. We propose that the transfer of Hf+ from formate to the active site base Y- is thermodynamically coupled to two-electron oxidation of the formate molecule, thereby facilitating formation of CO2. Under normal physiological conditions, when electron flow is not limited by the terminal acceptor of electrons, the energy released upon oxidation of Mo(IV) centers by the Fe4S4 is used for deprotonation of YHf and transfer of Hf+ against the thermodynamic potential.

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.

Quantitative kinetic model for photoassembly of the photosynthetic water oxidase from its inorganic constituents: requirements for manganese and calcium in the kinetically resolved steps,.

The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.

High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: proton equilibrium, cations, and electrostatics.

The kinetics of pulsed-light photoactivation, the light-induced reassembly of the water-oxidizing complex (WOC) of PSII in the presence of essential inorganic cofactors, has been studied using two improvements: a new efficient chelator, N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), for complete extraction of {Mn4} and Ca2+ and an ultrasensitive polarographic cell for O2 detection [Ananyev, G.M., & Dismukes, G.C. (1996) Biochemistry 35, 4102-4109]. Measurements have been made of the initial half-time, t1/2 (sum of the lag time for formation of the first intermediate, IM1, plus the half-time for formation of the second intermediate, IM2), and the steady-state yield, Yss, for recovery of O2 evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca2+, Mg2+, and Na+) slow the rate of photoactivation, even though Ca2+ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn(2+)-specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn2+ ions by electrostatic steering; (2) the Michaelis constant for the calcium requirement for Yss at sufficiently low Mn2+ concentrations (8 microM) that competition at the calcium site does not occur is K(m) = 1.4 mM. Numerically, K(m) is the same for reactivation of O2 evolution in Ca-depleted PSII membranes which retain four Mn ions; (3) in the absence of Ca2+ but in the presence of saturating amounts of Mn2+ (8 Mn/apo-WOC) and Cl-(35 mM) assembly of a stable tetra-Mn cluster occurs neither under illumination nor in the dark after subsequent addition of CaCl2. However, in the presence of suboptimal concentrations of calcium required for maximum Yss, calcium-dependent assembly of stable yet inactive clusters occurs in the light; (4) protons in equilibrium with the buffer greatly increase the half-time 3-fold between pH 6.75 and 5.4, indicating ionization of one or more protons from the first photo-oxidized intermediate formed prior to the rate-limiting step (photo-oxidation of the second Mn2+); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB-), a known reductant of intact WOC, increases the half-time 2.5-fold (< or = 40 microM) and paradoxically stimulates Yss by 50% at 20 microM concentration. These results suggest that TPB- increases the local concentration of Mn2+ adjacent to apo-WOC (Yss increase), while also reducing the S2 and S3 states of the intact WOC at higher concentrations (t1/2 increase). The effects of anions and cations indicates that overcoming the surface potential of the membrane/protein PSII complex may play an important role in the kinetics of reassembly of the {Mn4} cluster; (6) the ratio Y4/Y3 in the kinetics of O2 evolution from a series of single-turnover flashes, a ratio that typically reflects the probability of misses (alpha), grows noticeably larger with increasing extent of recovery of O2 evolving activity and also with increase in the amount of Mn2+, indicating competition between substrate water and excess Mn2+ for reduction of the functional {Mn4} cluster. On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H+ and Ca2+ in the formation of the first two intermediates.

The conformation of the isoprenyl chain relative to the semiquinone head in the primary electron acceptor (QA) of higher plant PSII (plastosemiquinone) differs from that in bacterial reaction centers (ubisemiquinone or menasemiquinone) by ca. 90 degrees.

The conformation and partial electron spin density distribution of the reduced primary electron acceptor (QA-), a plastosemiquinone-9 (PQ-9-) anion radical, in photosystem II protein complexes from spinach as well as free PQ-9- in solution have been determined by EPR and 1H ENDOR spectroscopies. The data show that the conformation of the isoprenyl chain at C beta relative to the aromatic ring differs by 90 degrees for QA- in higher plant PSII versus both types of bacterial reaction centers, Rhodobacter sphaeroides and Rhodopseudomonas viridis [containing ubiquinone (UQ) or menaquinone (MQ) at QA site, respectively]. This conformational distinction between the QA- species in PSII vs bacterial RCs follows precisely the conformational preferences of the isolated semiquinone anion radicals free in solution; type II semiquinones like PQ-9- have the isoprenyl C beta C gamma bond coplanar with the aromatic ring, while type I semiquinones like UQ- and MQ- place the C beta C gamma bond perpendicular to the ring. This conformational difference originates from nonbonded repulsions between the isoprenyl chain and the C6 methyl group present in type I semiquinones, forcing the perpendicular conformation, but absent in type II semiquinones having the smaller H atom at C6. Thus, the QA binding site in both higher plant PSII and bacterial reaction centers accommodates the lower energy conformation of their native semiquinones observed in solution. The energy difference between ground (C beta C gamma bond perpendicular to the ring) and excited (C beta C gamma bond coplanar with the ring) conformations of UQ- and vitamin K1- radicals is estimated to be sufficiently large (ca. 6 kcal/mol) to produce greater than a 10-fold difference in populations of these conformations at room temperature. For PQ-9-, a similar number is estimated. We propose that the strong confornational preferences of type I and type II semiquinones has lead to the evolution of different reaction center protein structures surrounding the isoprenyl/quinone head junction of QA to accommodate the favored low energy conformers. This predicted difference in protein structures could explain the low effectiveness (high selectivities) observed in quinone replacement experiments for type II vs type I quinones seen in higher plant PSII and bacterial reaction centers, respectively.

Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate.

The process of photoactivation, the assembly of the water-oxidizing complex (WOC) of photosystem II (PSII) membranes, has been examined using two major improvements in methodology. First a new lipophilic chelator, N,N,N',N'-tetrapropionate-1,3-bis(aminomethyl)benzene (TPDBA), has been used that permits complete extraction of both manganese and calcium and the three extrinsic WOC polypeptides while minimizing damage to the apo-PSII protein and, importantly, eliminating the need to use reductants. Second, an ultrasensitive, fast-response, polarographic cell and detection system were built. The apparatus features (a) an ultrabright red light-emitted diode (LED) for controlling the light intensity, pulse duration, and dark intervals, features critical for minimization of photoinhibition; (b) a microvolume (5 microL) O2 polarographic cell (Clark type) fitted with a thin silicone membrane for rapid response (100 ms); and (c) DC/AC preamplifier integrated into the microcell and interfaced to a bandpass AC amplifier. The sensitivity enables detection of approximately 5 x 10(-14) mol of O2 per flash at a signal to noise = 5/1. These improvements permit 100-fold lower Mn concentrations to be explored. Under optimum conditions, complete recovery of O2-evolving activity could be restored compared to that of PSII membranes depleted of the three extrinsic polypeptides (35% Vmax vs intact PSII). Titration of the photoactivation steady-state O2 yield, Yss, and the half-time for recovery, t1/2, vs Mn concentration demonstrate that 4.0 Mn/P680 are cooperatively taken up at 95% restoration of Yss and that 1.1-1.2 Mn atoms are involved in the rate-limiting photolytic step under steady-state conditions. Due to minimization of photoihibition, this intermediate exhibits a single exponential recovery kinetic over the entire population of PSII centers. Mn atoms in excess of 4 Mn/P680 accelerate the rate of photoactivation but decrease the yield above 8-10 Mn/P680. Maxima in both Yss and t1/2 are observed at similar electrochemical potentials of the medium, 380 and 340 mV, respectively. We attribute this maximum to either elimination of a recombination reaction between the redox-active tyrosine-161 of the D1 polypeptide (Y(Z)+) and an electron acceptor, possibly cytochrome b559, or stabilization of an intermediate in photoactivation. At low Mn2+ concentrations, a new pre-steady-state kinetic intermediate which binds fewer than 4 Mn atoms can be directly observed. This early kinetic phase has a rate that depends on Mn concentration and is independent of the electron acceptor identity and concentration.

Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ. and YD. in photosystem II using wild-type and the D2-Tyr160Phe mutant of Synechocystis 6803.

The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ. has been hampered by the simultaneous presence of the much more stable YD., the short lifetime of YZ., and the difficulty in trapping the YZ. radical at low temperature. We present here a method for obtaining an uncontaminated YZ. radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the beta-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ. EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ. radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ., this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ.. We have compared the environments of YZ. and YD. by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ. and YD. differ in line shape and line width. (2) Both YZ. and YD. exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD., indicating the hydrogen bond to be well-defined. In the case of YZ. it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD. possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 A. (4) All of the 1H couplings of YD. are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ. spectrum are broad. The environment surrounding YZ. appears to be more disordered and solvent-accessible.

Determination of the metal ion separation and energies of the three lowest electronic states of dimanganese (II,II) complexes and enzymes: catalase and liver arginase.

The dimanganese (II,II) catalase from Thermus thermophilus, MnCat(II,II), arginase from rat liver, Arg(II,II), and several dimanganese(II,II) compounds, LMn2XY2, which are functional catalase mimics, all possess a pair of coupled Mn(II) ions in their catalytic sites. For each of these, we have measured by EPR spectroscopy the relative energies separating the three lowest electronic states (singlet, triplet, and quintet), described a general method for extracting the individual spectra for these states by multicomponent analysis, and determined the Mn-Mn separation. The triplet-singlet and quintet-singlet energy gaps were modeled well by fitting the temperature dependence of the EPR intensities to a Boltzmann expression for a pair of Mn(II) ions coupled by isotropic Heisenberg spin exchange (-2JS1S2). This dependence indicates diamagnetic ground states with delta E10 (cm-1) = magnitude of 2J = 4 and 11.2 cm-1 for Arg-(II,II)(+borate) and MnCat(II,II)(phosphate), respectively. This large difference in magnitude of 2J reflects either a difference in the bridging ligands or, possibly, a weaker ligand field (larger ionization potential) for the Mn(II) ions in arginase. In n-butanol/CH2Cl2 the triplet-singlet energy gaps for [LMn2(CH3CO2)](C1O4)2 (1), [LMn2(CH3CO2)3] (2), and [LMn2Cl3] (3), where HL = N,N,N',N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan+ ++-2-ol, are 23-24 cm-1. Comparison of the Heisenberg exchange interaction constants for more than 30 dimanganese(II,II) complexes suggests a possible bridging structure of (mu-OH)(mu-carboxylate)1-2 for MnCat(II,II), while the 3-fold weaker coupling in Arg(II,II) suggests mu-aqua in place of mu-hydroxide. EPR spectra of both the triplet and quintet electronic states were extracted and found to exhibit zero-field splittings (ZFS) and resolved 55Mn hyperfine splittings indicating spin-coupled Mn2-(II,II) species. The major ZFS interaction could be attributed to the magnetic dipole-dipole interaction between the Mn(II) ions. A linear correlation is observed between the crystallographically determined Mn-Mn distance and the ZFS of the quintet state (D2) for five dimanganese pairs for which both data sets are available. Using this correlation, the Mn-Mn distance in Arg(II,II) is predicted to be 3.36-3.57 A for the native enzyme (multiple forms) and 3.59 A for MnCat(II,II)(phosphate). Addition of the inhibitor borate to Arg(II,II) simplifies the ZFS, indicative of conversion to a single species with mean Mn-Mn separation of 3.50 A. The second metal ion in dinuclear complexes possessing a shared bridging ligand has been shown to attenuate the strength of the mu-ligand field potential, as monitored by the strength of the single ion ZFS.(ABSTRACT TRUNCATED AT 250 WORDS)