Respiratory components in acidophilic bacteria that respire on iron

Abstract

Microorganisms capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Phylogenetically distinct organisms were shown to express spectrally distinct redox‐active biomolecules during autotrophic growth on soluble iron. A new iron‐oxidizing eubacterium, designated as strain Funis, was investigated. Strain Funis was judged to be different from other known iron‐oxidizing bacteria on the bases of comparative lipid analyses, 16S rRNA sequence analyses, and cytochrome composition studies. When grown autotrophically on ferrous ions, Funis produced conspicuous levels of a novel acid‐stable, acid‐soluble yellow cytochrome with a distinctive absorbance peak at 579 nm in the reduced state.

Stopped‐flow spectrophotometric kinetic studies were conducted on respiratory chain components isolated from cell‐free extracts of Thiobacillus ferrooxidans. Experimental results were consistent with a model where the primary oxidant of ferrous ions is a highly aggregated c‐type cytochrome that then reduces the periplasmic rusticyanin. The Fe(II)‐dependent, cytochrome c‐catalyzed reduction of the rusticyanin possessed three kinetic properties in common with corresponding intact cells that respire on iron: the same anion specificity, a similar dependence of the rate on the concentration of ferrous ions, and similar rates at saturating concentrations of ferrous ions     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1–100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Synthesis of a novel class of glycocluster with a cyclic α-(1→6)-octaglucoside as a scaffold and their binding abilities to concanavalin A

The synthesis of small glycoclusters with high affinity toward lectins is one of the important subjects in glycotechnology. Although cyclic α-(1→6)-d-octaglucoside (CI8) is an attractive scaffold on which to put glycosyl pendants, the compound has only secondary hydroxyl groups, which are relatively unreactive for substitution reactions. The oxidation of the vicinal diols of CI8 and reductive amination of the resultant dialdehydes with 2-aminoethyl mannoside gave mannose-CI8 conjugates with a variety of average mannose incorporation numbers (2-7). The average numbers were deduced from MALDI-TOF mass and ¹H NMR spectroscopy. The binding ability of mannose-CI8 conjugates to concanavalin A increased with the increasing numbers of average mannose incorporation, reaching a plateau at tetravalence, as estimated from a latex bead-based agglutination lectin assay. Toxicity tests demonstrated the biocompatibility of mannose-CI8 conjugates.     

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

Subcellular distribution,molecular dynamics and catabolism of plasmalogens in myocardium

Recent studies have implicated accelerated sarcolemmal phospholipid catabolism as a mediator of the lethal sequelae of atherosclerotic heart disease. We have demonstrated that plasmalogens are the predominant phospholipid constituents of canine myocardium and that plasmalogens are hydrolyzed by a novel calcium independent plasmalogen selective phospholipase A2. Since the activities of phospholipases are modulated by the molecular dynamics and interfacial characteristics of their phospholipid substrates, we compared the molecular dynamics of plasmenylcholine and phosphatidylcholine vesicles by electron spin resonance spectroscopy and deuterium magnetic resonance spectroscopy. Plasmenylcholine vesicles have separate and distinct molecular dynamics in comparisons to their phosphatidylcholine counterparts as ascertained by substantial decreases in the angular fluctuations and motional velocities of probes attached to their sn-2 aliphatic constituents. Furthermore, since free radical oxidation of myocardial lipid constituents occurs during myocardial ischemia and reperfusion, we demonstrated that ¹O₂ mediated oxidation of plasmenylcholine resulted in the generation of several products which have chromatographic characteristics and molecular masses corresponding to 2-acyl lysophosphatide derivatives. Taken together, these studies underscore the biologic significance of the predominance of sarcolemmal plasmalogens present in mammalian myocardium and suggest that their catabolism by plasmalogen selective phospholipases and/or oxidative processes may contribute to the lethal sequelae of myocardial ischemia.     

Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.     

Measurement of adrenolutin as an oxidation product of catecholamines in plasma

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (M) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed, into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of ³H-adrenolutin after injection of ³H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Formation of Metallogenium-like structures by a manganese-oxiding fungus

Structures resembling Metallogenium spp. were observed in agar and in liquid cultures of a Mn-oxidizing basidiomycetous fungus only when Mn²⁺ was oxidized. Fungal viability was necessary for formation of the structures; Mn²⁺ concentration and the presence or absence of agar in the medium were important factors determining their morphology. Slide cultures revealed no identifiable cells in any stage of development. Fluorescent dyes that stained nucleic acids and polysaccharides in the fungal hyphae did not stain the Metallogenium-like structures. Likewise, Rhodamine 123, a fluorescent probe for membrane potential, stained fungal mitochondria, but did not stain the structures. Thin sections through the structures showed no biological membranes or other cellular features. Only the characteristic ultrastructure of biological Mn oxides were observed in serial thin sections. In agar, unfixed structures disappeared permanently during reduction of Mn oxides with hydroxylamine. Glutaraldehyde fixation stabilized these structures. Fixed structures lost most of their original phase density during reduction with hydroxylamine, but continuous microscopic observations showed that their phase density could be restored by staining with Coomassie blue. Structures that formed in liquid medium did not require stabilization with glutaraldehyde during reduction of Mn oxides. They, too, lost their original phase density during reduction with hydroxylamine; phase density could be restored by staining with cationic colloidal iron or Coomassie blue. The results suggest that the Metallogenium-like structures were formed as a result of Mn oxidation associated with exopolymers produced by the fungus.Non-standard abbreviations HEPES (N-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - DAPI (4,6-diamidino-2-phenylindole) - PIPES (piperazine-N,N-bis[2-ethane sulfonic acid])