A nhaD Na/Hantiporter and a pcd homologues are among the Rhodothermus marinus complex I genes

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo₁ to nqo₇ (nqo_A) and nqo₁₀ to nqo₁₄ (nqo_B)] and two independent genes (nqo₈ and nqo₉). Unexpectedly, two genes encoding homologues of a NhaD Na⁺/H⁺ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqo_B, flanked by nqo₁₃ and nqo₁₄. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqo_B. The possible role of these two genes in R. marinus complex I is discussed.     

The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc₁ complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa₃ oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa₃ oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.     

Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc1 complex

Previous studies indicate that the three-subunit cytochrome bc₁ core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.     

Solute carrier family 26 member a2 (Slc26a2) protein functions as an electroneutral SOFormula/OH-/Cl- exchanger regulated by extracellular Cl-

Slc26a2 is a ubiquitously expressed SO(4)(2-) transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO(4)(2-) and the ion gradients that mediate SO(4)(2-) uptake are poorly understood. We report here that Slc26a2 functions as an SO(4)(2-)/2OH(-), SO(4)(2-)/2Cl(-), and SO(4)(2-)/OH(-)/Cl(-) exchanger, depending on the Cl(-) and OH(-) gradients. At inward Cl(-) and outward pH gradients (high Cl(-)(o) and low pH(o)) Slc26a2 functions primarily as an SO(4)(2-)(o)/2OH(-)(i) exchanger. At low Cl(-)(o) and high pH(o) Slc26a2 functions increasingly as an SO(4)(2-)(o)/2Cl(-)(i) exchanger. The reverse is observed for SO(4)(2-)(i)/2OH(-)(o) and SO(4)(2-)(i)/2Cl(-)(o) exchange. Slc26a2 also exchanges Cl(-) for I(-), Br(-), and NO(3)(-) and Cl(-)(o) competes with SO(4)(2-) on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO(4)(2-)(i)/2OH(-)(o) exchange. Slc26a2 can transport oxalate in exchange for OH(-) and/or Cl(-) with properties similar to SO(4)(2-) transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence (367)GFXXP(371) between TMD7 and TMD8 close to the conserved Glu(417) in the permeation pathway. Mutation of Glu(417) eliminated transport by Slc26a2, whereas mutation of Phe(368) increased the affinity for SO(4)(2-)(o) 8-fold while reducing the affinity for Cl(-)(o) 2 fold, but without affecting regulation by Cl(-)(o). These findings clarify the mechanism of net SO(4)(2-) transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.     

Dual inhibitory effect of Glycyrrhiza glabra (GutGard™) on COX and LOX products

Glycyrrhiza glabra and its phytoconstituents have been known to possess widespread pharmacological properties as an anti-inflammatory, anti-viral, antitumour and hepatoprotective drug. In this study, we examined the inhibitory potential of extract of G. glabra (GutGard™) root and its phytoconstituents (glabridin, glycyrrhizin, and isoliquiritigenin) on both cyclooxygenase (COX) and lipoxygenase (LOX) products in order to understand the mechanism of its anti-inflammatory action. Inhibitory effect of GutGard™ and its phytoconstituents on lipopolysaccharide (LPS) induced prostaglandin E₂ (PGE₂), calcimycin (A23187) induced thromboxane (TXB₂), and leukotriene (LTB₄) release was studied using murine macrophages (J774A.1) and human neutrophil (HL-60) cells. Results revealed that, G. glabra and glabridin significantly inhibited PGE₂, TXB₂ (COX) and LTB₄ (LOX), while, isoliquiritigenin exerted inhibitory effect only against COX products but failed to suppress LOX product. However, glycyrrhizin at the tested concentrations failed to exhibit inhibitory effect on both COX and LOX products. Here, we report for the first time that G. glabra (almost devoid of glycyrrhizin) exhibits anti-inflammatory property likely through the inhibition of PGE₂, TXB₂ and LTB₄ in mammalian cell assay system, which could be influenced in part by glabridin and isoliquiritigenin.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.     

A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase

Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.     

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.     

Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury

To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca²⁺. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle.