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     

Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.     

Fluorometric, Chromatographic, and Spectronic Evidence for the Non-indolic Nature of Citrus Auxin

Evidence for the non-indolic nature of the new citrus auxinis presented on the basis of fluorometric properties, thin-layerchromatography, Ehrlich's colour reaction, paper electrophoresis,and the infra-red spectra determinations. Citrus auxin had alower Rf in TLC than IAA, did not give the typical indole reactionwith Ehrlich's reagent, and behaved differently in electrophoresis.The infra-red spectra also provided preliminary informationconcerning chemical structure. The hypothesis that indolic compoundsconstitute the only natural auxins in higher plants should berevised in view of this evidence that a non-indole auxin existsin higher plants.     

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for NH2 group rotation in buried asparagine side chains

Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.     

Assignment of asparagine-44 side-chain primary amide 1H NMR resonances and the peptide amide N1H resonance of glycine-37 in basic pancreatic trypsin inhibitor

New assignments of three previously undetected amide proton NMR resonance lines in bovine pancreatic trypsin inhibitor are reported. These are the peptide amide proton of Gly-37 and the primary amide protons of Asn-44. Specific assignments of Asn-44 and Asn-43 HE and HZ resonances are also reported. The Gly-37 NH and Asn-44 HZ resonances are shifted upfield to 4.3 and 3.4 ppm, respectively, by the ring current of the Tyr-35 aromatic group, while Asn-44 HE resonates at 7.8 ppm. The abnormal chemical shifts of Asn-44 HZ and Gly-37 NH indicate that both NH's interact with the pi-electron cloud of the Tyr-35 ring. This is consistent with their location in the crystal structure. The resonances are resolved by differential labeling techniques and are studied by combined use of NOE and exchange difference spectroscopy.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.     

Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.