A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by Y_Z. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[−Mn]) membranes. The rate of Mn(II) oxidation saturates at ≥10 μM in PSII(−Mn) membranes and ≥500 μM in EDC-treated PSII (−Mn) samples. Intact PSII(−Mn) membranes have only one site for Mn(II) oxidation via Y_Z (dissociation constant, K_d = 0.64 μM), while EDC-treated PSII(−Mn) samples have two sites (K_d = 1.52 and 22 μM; the latter is the low-affinity site). When PSII(−Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (K_d = 2.3 μM) for exogenous Mn(II) oxidation by Y_Z radical. In this case, the rate of electron donation via Y_Z saturated at a Mn(II) concentration ≥15 μM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HA_Z site is protected from the action of the modifier by the iron cations blocking the HA_Z site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HA_Z site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

Low-barrier hydrogen bond plays key role in active photosystem II — A new model for photosynthetic water oxidation

The function and mechanism of Tyr_Z in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr_Z, and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr_Z function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr_Z oxidation and Tyr_Z^· reduction during photosynthetic water oxidation. Upon the oxidation of Tyr_Z, the hydrogen bond between Tyr_Z and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr_Z^· reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr_Z oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr_Z oxidation and Tyr_Z^· reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr_Z and Tyr_D in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr_Z. This type of change arising from radiation damage has been confirmed in other enzyme systems.     

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

Low-temperature electron transfer suggests two types of QA in intact photosystem II

The correlation between the reduction of Q_A and the oxidation of Tyr_Z or Car/Chl_Z/Cyt_b559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g = 1.95-1.86 Q_A^-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q_A^-•, Car^+•/Chl^+• and oxidized Cyt_b559, while enhancing the amplitude of the S₁Tyr_Z• EPR signal in the intact PSII sample. The quantification and stability of the g = 1.95-1.86 EPR signal and signals arising from the oxidized Tyr_Z and the side-path electron donors, respectively, indicate that the EPR-detectable g = 1.95-1.86 Q_A^-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl_Z/Cyt_b559), but not of Tyr_Z. These results imply that two types of Q_A^-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q_A are discussed.     

Photo-oxidation of tyrosine in a bio-engineered bacterioferritin ‘reaction centre’—A protein model for artificial photosynthesis

The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Manganese(III) complexes of N2O2 donor 5-bromosalicylideneimine ligands: Combined effects of electron withdrawing substituents and chelate ring size variations on electrochemical properties

A series of mononuclear manganese(III) complexes of formulae [Mn(L)(X)(H₂O)] (1-13) and [Mn(L)(X)] (14-17) (X = ClO₄, F, Cl, Br, I, NCS, N₃), derived from the Schiff bases of 5-bromosalicylaldehyde and different types of diamine (1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane and 1,4-diaminobutane), have been synthesized and characterized by the combination of IR, UV-Vis spectroscopies, cyclic voltammetry and by X-ray crystallography. The redox properties of all the manganese(III) complexes show grossly identical features consisting of a reversible or quasireversible Mn^III/Mn^II reduction. Besides Mn^III/Mn^II reduction, the complexes 4, 5, 10, 13 and 16 also show reversible or quasireversible Mn^III/Mn^IV oxidation. A linear correlation has been found for the complexes 5, 7, 11 and 13 [Mn(L²)(X)(H₂O)] (X = F, Cl, Br, I) when E_1/2 [Mn^III/Mn^II] is plotted against Mulliken electronegativities (χ_M). The effect of the flexibility of the ligand on redox potential has been studied. It has been observed that the manganese(II) state is stabilized with increasing flexibility of the ligand environment. The crystal structure of 6 shows an octahedral geometry.     

Ultrahigh (0.93 Å) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: Implications for the catalytic mechanism

Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn^II to Mn^III. The Mn^III is released from the enzyme in complex with oxalate, enabling the oxalate-Mn^III complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn^II ligands in our previous crystal structures of native MnP. In our current work, new 0.93 Å and 1.05 Å crystal structures of MnP with and without bound Mn^II, respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93 Å resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Ångstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.     

Molecular interference of Cd with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd²⁺ is known to exchange, with high affinity in a slow reaction, for the Ca²⁺ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd²⁺ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd²⁺-binding to those sites, we have studied how Cd²⁺ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd²⁺ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd²⁺ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y_Z to P₆₈₀⁺ was inhibited; (ii) Q_A⁻ to Q_B electron transfer was slowed down; (iii) the S₂ state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q_A⁻Fe²⁺ was modified but its amplitude was not sensitive to the presence of Cd²⁺. In addition, the presence of both Ca²⁺ and DCMU abolished Cd²⁺-induced effects partially and in different sites. The number of sites for Cd²⁺ binding and the possible nature of these sites are discussed.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Bioinspired FeIIICdII and FeIIIHgII complexes: synthesis, characterization and promiscuous catalytic activity evaluation

In this work we report on the synthesis, crystal structure, and physicochemical characterization of the novel dinuclear [Fe^IIICd^II(L)(μ-OAc)₂]ClO₄·0.5H₂O (1) complex containing the unsymmetrical ligand H₂L = 2-bis[{(2-pyridyl-methyl)-aminomethyl}-6-{(2-hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methylphenol. Also, with this ligand, the tetranuclear [Fe₂^IIIHg₂^II(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) and [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) complexes were synthesized and fully characterized. It is demonstrated that the precursor [Fe^III₂Hg^II₂(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) can be converted to (3) by the fixation of atmospheric CO₂ since the crystal structure of the tetranuclear organometallic complex [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) with an unprecedented {Fe^III(μ-O_phenoxo)₂(μ-CO₃)Fe^III} core was obtained through X-ray crystallography. In the reaction 2 → 3 a nucleophilic attack of a Fe^III-bound hydroxo group on the CO₂ molecule is proposed. In addition, it is also demonstrated that complex (3) can regenerate complex (2) in aqueous/MeOH/NaOH solution. Magnetochemical studies reveal that the Fe^III centers in 3 are antiferromagnetically coupled (J = − 7.2 cm^− 1) and that the Fe^III-OR-Fe^III angle has no noticeable influence in the exchange coupling. Phosphatase-like activity studies in the hydrolysis of the model substrate bis(2,4-dinitrophenyl) phosphate (2,4-bdnpp) by 1 and 2 show Michaelis-Menten behavior with 1 being ~ 2.5 times more active than 2. In combination with k_H/k_D isotope effects, the kinetic studies suggest a mechanism in which a terminal Fe^III-bound hydroxide is the hydrolysis-initiating nucleophilic catalyst for 1 and 2. Based on the crystal structures of 1 and 3, it is assumed that the relatively long Fe^III…Hg^II distance could be responsible for the lower catalytic effectiveness of 2.     

Stepwise Transition of the Tetra-Manganese Complex of Photosystem II to a Binuclear Mn2(μ-O)2 Complex in Response to a Temperature Jump: A Time-Resolved Structural Investigation Employing X-Ray Absorption Spectroscopy

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25°C to 47°C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y_D^ox radical and of released Mn²⁺ by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y_D^ox occur simultaneously (k₁ = 1.0 min⁻¹). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by ~3.3 Å, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by ~2.7 Å in the native complex are reduced to Mn(II) and released (k₂ = 0.18 min⁻¹). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di-μ-oxo bridged Mn(III)₂ complex. (3) Finally, the tightly-bound Mn₂(μ-O)₂ unit is slowly reduced and released (k₃ = 0.014 min⁻¹).     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine_Z (Tyr_Z) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr_Z with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr_Z and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr_Z oxidation at cryogenic temperature in the S₀ and S₁ states was around neutral pH and was essentially pH-independent. The yield of Tyr_Z oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr_Z^· in the S₀ and S₁ states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr_Z oxidation and Tyr_Z^· reduction at cryogenic temperature in the S₀ and S₁ states of the active PSII. Theoretical calculations indicate that Tyr_Z becomes more difficult to oxidize when a H₂O molecule interacts directly with it. It is suggested that Tyr_Z is probably located in a hydrophobic environment with no direct interaction with the substrate H₂O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.     

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, Y_Z. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O₂ as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of Y_Z by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking Y_Z by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680⁺ under the conditions of the Y_Z blockade by Fe cations. Neither an alternative donor for P680, Y_D , nor cytochrome b ₅₅₉ participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 12–20.Original Russian Text Copyright © 2005 by Lovyagina, Davletshina, Kultysheva, Timofeev, Ivanov, Semin.