Biochemical and biophysical studies on cytochrome aa3. VII. The effect of cytochrome c on the oxidation-reduction potential of isolated cytochrome aa3

1.

1. In the absence of cytochrome c, the haem groups of cytochrome aa₃ titrate as indistinguishable identities, each having E′_o = 280 mV and n = 1.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.     

DFT study of nano zinc/copper voltaic cells

To facilitate the development of new materials for use in batteries, it is necessary to develop ab initio full-electron computational techniques for modeling potential new battery materials. Here, we tested density functional theory procedures that are accurate enough to obtain the energetics of a zinc/copper voltaic cell. We found the magnitude of the zero-point energy correction to be 0.01–0.2 kcal/mol per atom or molecule and the magnitude of the dispersion correction to be 0.1–0.6 kcal/mol per atom or molecule for Zn _n , (H₂O) _n , \( \mathrm{Zn}{\left({\mathrm{H}}_2\mathrm{O}\right)}_n^{2+} \), \( \mathrm{Cu}{\left({\mathrm{H}}_2\mathrm{O}\right)}_n^{2+} \), and Cu _n . Counterpoise correction significantly affected the values of ?\( {E}_n^{\mathrm{abs}} \), ?\( {E}_n^{\mathrm{coh}} \), and ?E^solv by 1.0–3.1 kcal/mol per atom or molecule at the B3PW91/6-31G(d) level of theory, but by only 0.04–0.4 kcal/mol per atom or molecule at the B3PW91/cc-pVTZ level of theory. The application of B3PW91/6-31G(d) yielded results that differed from macroscopic experimental values by 0.1–7.1 kcal/mol per atom or molecule, whereas applying B3PW91/cc-pVTZ produced results that differed from macroscopic experimental values by 0.1–4.8 kcal/mol per atom or molecule, with the smallest differences occurring for reactions with a small macroscopic experimental ?E and the largest differences occurring for reactions with a large macroscopic experimental ?E, implying size consistency.     

Coordination chemistry of anticrowns: Synthesis and X-ray crystal structure determination of the polydecker sandwich complexes of cyclic trimeric perfluoro-o-phenylenemercury with azulene and 6-(N,N-dimethylamino)pentafulvene

The interaction of cyclic trimeric perfluoro-o-phenylenemercury (o-C₆F₄Hg)₃ (1) with azulene results in the formation of a complex, {[(o-C₆F₄Hg)₃](azulene)} (2), containing one molecule of azulene per one macrocycle molecule. The complex represents a polydecker sandwich wherein the azulene units alternate with the molecules of the mercury anticrown. The reaction of 1 with 6-(N,N-dimethylamino)pentafulvene (DMAPF) also gives a 1:1 complex, {[(o-C₆F₄Hg)₃](DMAPF)} (3), having a polydecker sandwich structure in the crystal. In complex 2, both the C₅ and C₇ rings of the azulene ligand are involved in the bonding to the Hg sites of 1. In complex 3, the C₅ ring of the fulvene ligand together with its exocyclic carbon atom take part in the coordination to the mercury centres. In both adducts, the negatively charged five-membered ring of the azulene and, correspondingly, the fulvene moieties is arranged in the space between the central Hg₃C₆ rings of the adjacent macrocycles while the remaining portion of these moieties is disposed outside this space. The molecules of azulene and DMAPF in 2 and 3 are bonded to 1 through donation of their π-electrons on vacant orbitals of the Hg atoms. The synthesized 2 and 3 are the first examples of structurally characterized complexes of azulene and DMAPF with a non-transition metal compound.     

The peculiarities of the structural and functional state of the cytochrome component of the liver mitochondrial respiratory chain under conditions of acetaminophen-induced hepatitis on the background of alimentary protein deprivation

The activity of a key enzyme of the cytochrome component of the respiratory chain (cytochrome oxidase), the quantitative redistribution of mitochondrial cytochromes b, c ₁, c, and aa ₃, as well as the activities of the key enzymes of cytochrome heme metabolism (δ-aminolevulinate synthase and heme oxygenase) under conditions of acetaminophen-induced liver injury were studied on the background of dietary protein deprivation. Under conditions of acetaminophen-induced hepatitis that developed on the background of alimentary protein deprivation, an inhibition of cytochrome oxidase activity and a decrease in the contents of mitochondrial cytochromes on the background of an increase in the δ-aminolevulinate synthase and heme oxygenase activity were observed. In animals with a toxic liver injury that were kept under conditions of dietary protein deprivation, the contents of mitochondrial cytochromes b, c ₁, c, and aa ₃ progressively decreased, which was accompanied by an increase in heme oxygenase activity, whereas δ-aminolevulinate synthase activity remained at the control level. It was concluded that dietary protein deprivation is a critical factor for the development of disturbances in the structural-functional integrity of the cytochrome component of the respiratory chain. The identified changes can be considered as a possible mechanism that underlies the disturbance in the function of the energy biotransformation system under conditions of dietary protein deprivation.     

Ferredoxin-glutamate synthase from Chlamydomonas reinhardii. Prosthetic groups and preliminary studies of mechanism

• 1.1. Ferredoxin-glutamate synthase from the green alga Chlamydomonas reinhardii appears to contain as prosthetic groups, 1 FAD, 1 FMN and 1 |3Fe-xS | cluster, per molecule of M_r = 146,000.
• 2.2. The synthesis of glutamate, catalyzed by this enzyme, proceeds through the formation of an enzyme-bound free radical of flavin semiquinone.
• 3.3. This enzyme may catalyze the ferredoxin-dependent reduction of 2-oxoglutarate, in the absence of glutamine.

     

Evidence for the involvement of non-heme iron in the active site of hydrogenase from Desulfovibrio vulgaris

1.

1. Hydrogenase (hydrogen: cytochrome c₃ oxidoreductase) of high specific activity has been purified to near homogeneity as judged by disc electrophoresis and in the analytical ultracentrifuge, and found to contain 3.5 iron and 3.2 labile-sulfide atoms per molecule (of mol. wt. 60 000).     

Comparative and evolutionary analysis of α-amylase gene across monocots and dicots

α-amylase is an important enzyme involved in starch degradation to provide energy to the germinating seedling. The present study was conducted to reveal structural and functional evolution of this gene among higher plants. Discounting polyploidy, most plant species showed only a single copy of the gene making multiple isoforms in different tissues and developmental stages. Genomic length of the gene ranged from 1472 bp in wheat to 2369 bp in soybean, and the size variation was mainly due to differences in the number and size of introns. In spite of this variation, the intron phase distribution and insertion sites were mostly conserved. The predicted protein size ranged from 414 amino acid (aa) in soybean to 449aa in Brachypodium. Overall, the protein sequence similarity among orthologs ranged from 56.4 to 97.4 %. Key motifs and domains along with their relative distances were conserved among plants although several species, genera, and class specific motifs were identified. The glycosyl hydrolase superfamily domain length varied from 342aa in soybean to 384aa in maize and sorghum while length of the C-terminal β-sheet domain was highly conserved with 61aa in all monocots and Arabidopsis but was 59aa in soybean and Medicago. Compared to rice, 3D structure of the proteins showed 89.8 to 91.3 % similarity among the monocots and 72.7 to 75.8 % among the dicots. Sequence and relative location of the five key aa required for the ligand binding were highly conserved in all species except rice.     

Transient Transcriptional Regulation of the CYS-C1 Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response

Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system.The gaseous hormone ethylene is known to regulate multiple physiological and developmental processes in plants, such as seedling emergence, leaf and flower senescence, climacteric fruit ripening, and organ abscission. Ethylene is also involved in the response of plants to abiotic and biotic stresses (Wang et al., 2002; Broekaert et al., 2006; van Loon et al., 2006). Enhanced ethylene production is an early, active response of plants to the perception of pathogen attack and is associated with the induction of defense reactions. During ethylene biosynthesis, S-adenosyl-l-Met is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. ACC is finally oxidized by ACC oxidase to form ethylene, carbon dioxide, and cyanide (Hartley et al., 1998; Wang et al., 2002). Hydrogen cyanide is a colorless and highly volatile liquid. The anion cyanide is toxic and renders the cells of an organism unable to use oxygen, primarily through the chelation of divalent and trivalent metal ions in the prosthetic groups of several metalloenzymes, including copper/zinc superoxide dismutase, catalase, nitrate and nitrite reductase, nitrogenase, peroxidases, and the mitochondrial cytochrome c oxidase (Isom and Way, 1984; Donato et al., 2007).Cyanide must be rapidly detoxified and metabolized by the plant to keep the concentration below toxic levels. Plants detoxify cyanide primarily through the enzyme β-cyanoalanine synthase (CAS), for which considerable levels of activity are constitutively found in many plant species. Rhodanese and mercaptopyruvate sulfurtransferase activities also make minor contributions to the cyanide detoxification process (Miller and Conn, 1980). CAS is a pyridoxal phosphate-dependent enzyme that converts Cys and cyanide to hydrogen sulfide and β-cyanoalanine, which is later converted to Asn, Asp, and ammonia by NIT4 class nitrilases (Piotrowski, 2008). Arabidopsis (Arabidopsis thaliana) plants carry the mitochondrial CAS CYS-C1 (At3g61440; Watanabe et al., 2008), which belongs to the family of β-substituted Ala synthase enzymes. The family also includes the three major O-acetyl-serine(thiol)lyase enzymes OAS-A1 (At4g14880), OAS-B (At2g43750), and OAS-C (At3g59760; Watanabe et al., 2008), the l-Cys desulfhydrase DES1 (At5g28030; Álvarez et al., 2010), the S-sulfocysteine synthase CS26 (At3g03630; Bermúdez et al., 2010), and the functionally unknown cytosolic isoforms CYS-D1 (At3g04940) and CYS-D2 (At5g28020). Mutations in CYS-C1 result in plants that accumulate cyanide and that display abnormal root hair (García et al., 2010), suggesting that cyanide has a signaling role in root development. The lack of the mitochondrial O-acetyl-serine(thiol)lyase isoform OAS-C, which is necessary to detoxify the sulfide released by the CAS activity, causes an accumulation of sulfide and cyanide and a root phenotype similar to the cys-c1 loss-of-function mutant (Álvarez et al., 2012b).Several authors have suggested that cyanide could act as a regulator of other metabolic processes in addition to performing the described role in plant root development (Siegien and Bogatek, 2006). It has been observed that this molecule is released during seed germination and that exogenously applied hydrogen cyanide breaks seed dormancy in several plants (Cohn and Hughes, 1986; Fol et al., 1989; Bogatek et al., 1991; Bethke et al., 2006). The role of cyanide as a regulatory molecule is not restricted to plants, and it has been demonstrated that cyanide is generated in leukocytes from Gly via a peroxidase (Stelmaszyńska, 1986) as well as in the central nervous system, where it has been hypothesized to act as a neuromodulator (Gunasekar et al., 2000; Cipollone and Visca, 2007). Cyanide production can be stimulated by opiates and decreased by treatment with muscarinic receptor agonists (Borowitz et al., 1997; Gunasekar et al., 2004).Despite the variety of known functions for cyanide in different organisms, the role of cyanide production in plants seems to have been unevaluated to date. In cyanogenic plants, cyanide is produced during the degradation of cyanogenic lipids and from the catabolism of cyanogenic glycosides (Poulton, 1990). Cyanide and cyanogenic compounds play an important role in plant defense against herbivores (Zagrobelny et al., 2008). In noncyanogenic plants, cyanide is a coproduct of ethylene biosynthesis. The molecule is also produced during the biosynthesis of camalexin, a phytoalexin formed in Arabidopsis plants upon infection by a large variety of microorganisms, including bacteria, fungi, and oomycetes (Glawischnig, 2007). During camalexin biosynthesis, the Trp-derived intermediate indole-3-acetonitrile is conjugated with Cys and serves as a substrate for the cytochrome P450 enzyme CYP71B15. This enzyme catalyzes the formation of the thiazoline ring as well as the release of cyanide and subsequent oxidative decarboxylation of dihydrocamalexic acid to camalexin (Glawischnig, 2007; Böttcher et al., 2009). Since both cyanide sources, camalexin and ethylene, are produced after pathogen attack, cyanide should be produced at significant levels during the plant response to pathogens. It has been shown that exogenous cyanide can enhance the resistance of tobacco (Nicotiana tabacum) and Arabidopsis leaves to Tobacco mosaic virus and Turnip vein clearing virus, respectively (Chivasa and Carr, 1998; Wong et al., 2002). Recently, it has been demonstrated that exogenously applied cyanide increases the resistance of young rice (Oryza sativa) plants to blast fungus infection, suggesting that cyanide rather than ethylene contributes to plant resistance (Seo et al., 2011).This work aims to further investigate the role of endogenously produced cyanide in the plant immune response by analyzing the behavior of Arabidopsis knockout mutants of the mitochondrial CAS CYS-C1 and the regulation of CYS-C1 in response to pathogen attack.     

Supramolecular assemblies of new 2-D complexes: Syntheses, crystal structures, spectroscopic and magnetic properties of {[MnAu2(CN)4(NITpPy)2(H2O)2]}n and {[Co(N(CN)2)2(NITpPy)2(H2O)2]}n

Two new complexes, {[MnAu₂(CN)₄(NITpPy)₂(H₂O)₂]}_n (1) and {[Co(N(CN)₂)₂(NITpPy)₂(H₂O)₂]}_n (2), have been synthesized and characterized. The single-crystal X-ray analysis for the complexes 1 and 2 demonstrates that each M(II) (M = Mn or Co) ion assumes a distorted octahedral MN₄O₂ coordination polyhedron. Four nitrogen atoms come from the cyanide groups and the pyridyl rings in a common plane, and two oxygen atoms come from the H₂O molecules in trans-positions. The structures of complexes 1 and 2 illustrate that aurophilicity and/or hydrogen bonding interactions play important roles in increasing dimensionality. Magnetic investigations on complexes 1 and 2 show the presence of weak antiferromagnetic interactions.     

Interactions of thiamine monophosphate (TMP) with one- and two-dimensional polymeric halogenomercurate anions. Crystal structures of (TMP)(Hg2Br5)·0.5H2O and (TMP)2(Hg3I8)

An investigation into the interactions between thiamine monophosphate (TMP) and anions has resulted in the preparation and X-ray characterization of the compounds (TMP)(Hg₂Br₅)·0.5H₂O (1) and (TMP)₂(Hg₃I₈) (2). In each compound the TMP molecule exists as a monovalent cation in the usual F conformation. The halogenomercurate anions occur in two-dimensional (2-D) network in 1 or one-dimensional (1-D) chain in 2. In both 1 and 2, the structures consist of alternating cationic sheets of the hydrogen-bonded TMP molecules and anionic sheets of the polymeric halogenomercurate anions. The TMP molecule binds to the polymeric anions through the characteristic ‘anion bridge I’, C(2)H?X?pyrimidinium (X=Br in 1 and I in 2), and electrostatic interactions between electropositive S(1) and halogen atoms. The ‘anion bridge II’ of the type N(4′1)H?X?thiazolium (X=phosphate group) plays a role in stabilizing the molecular conformation. The biological implication of the host-guest-like complexation between TMP and polymeric anions is discussed.     

Self-assembly of three hetero- and homopolynuclear cyanide bridged complexes of Fe-Cu and Cu: hydrothermal syntheses, structural characterization and properties

Reaction of Cu^II, K₃ [Fe(CN)₆] and bidentate diimine ligands by hydrothermal synthesis under different conditions affords one novel heteronuclear Fe^II-Cu^I complex, (bipy = 2,2′-bipyridine), and two homonuclear Cu^I complexes, [Cu^I(μ-CN)(bipy)]_n (2) and (3) (phen = 1,10-phenanthroline). Although all the three complexes are 1D cyanide bridged helical chains, they have different helicoids of pseudo-square, pseudo-trigonal and head-to-head bistrigonal for 1, 2 and 3, respectively. The structure of 1 is extended to 2D hexagonal meshed layers by the hydrogen bonding between terminal cyanides and lattice water molecules, which also contain π-π interactions between adjacent sheets. Cu^I ions in 1 are distorted trigonal planar coordinated by two bridging cyanides and one terminal cyanide, whereas that in 2 and 3 are pseudo-tetrahedral coordinated by two bridging cyanides and two N atoms of a diimine. Both the latter homometallic polymers exhibit similar chain structure, and these chains are close packed with their six adjacent chains in a parallel fashion along the c-axis to form a honeycomb network. It should be noted that complex 1 is the first cyanide bridged Fe^II-Cu^I complex of helical chain structure. The spectroscopic properties of complexes 1-3 have also been investigated.