Peter M. H. Kroneck

Tissue- and age-dependent differences in the complexation of cadmium and zinc in the cadmium/zinc hyperaccumulator Thlaspi caerulescens (Ganges ecotype) revealed by x-ray absorption spectroscopy.

Extended x-ray absorption fine structure measurements were performed on frozen hydrated samples of the cadmium (Cd)/zinc (Zn) hyperaccumulator Thlaspi caerulescens (Ganges ecotype) after 6 months of Zn2+ treatment with and without addition of Cd2+. Ligands depended on the metal and the function and age of the plant tissue. In mature and senescent leaves, oxygen ligands dominated. This result combined with earlier knowledge about metal compartmentation indicates that the plants prefer to detoxify hyperaccumulated metals by pumping them into vacuoles rather than to synthesize metal specific ligands. In young and mature tissues (leaves, petioles, and stems), a higher percentage of Cd was bound by sulfur (S) ligands (e.g. phytochelatins) than in senescent tissues. This may indicate that young tissues require strong ligands for metal detoxification in addition to the detoxification by sequestration in the epidermal vacuoles. Alternatively, it may reflect the known smaller proportion of epidermal metal sequestration in younger tissues, combined with a constant and high proportion of S ligands in the mesophyll. In stems, a higher proportion of Cd was coordinated by S ligands and of Zn by histidine, compared with leaves of the same age. This may suggest that metals are transported as stable complexes or that the vacuolar oxygen coordination of the metals is, like in leaves, mainly found in the epidermis. The epidermis constitutes a larger percentage of the total volume in leaves than in stems and petioles. Zn-S interaction was never observed, confirming earlier results that S ligands are not involved in Zn resistance of hyperaccumulator plants.

Structure of cytochrome c nitrite reductase

The enzyme cytochrome c nitrite reductase catalyses the six-electron reduction of nitrite to ammonia as one of the key stepsin the biological nitrogen cycle, where it participates inthe anaerobic energy metabolism of dissimilatory nitrate ammonification. Here we report on the crystal structure of this enzyme from the microorganism Sulfurospirillum deleyianum, which we solved by multiwavelength anomalous dispersion methods. We propose a reaction scheme for the transformation of nitrite based on structural and spectroscopic information. Cytochrome c nitrite reductase is a functional dimer, with 10 close-packed haem groups of type c and an unusual lysine-coordinated high-spin haem at the active site. By comparing the haem arrangement of this nitrite reductase with that of other multihaem cytochromes, we have been able to identify a family of proteins in which the orientation of haem groups is conserved whereas structure and function are not.

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.

Respiratory transformation of nitrous oxide (N2O) to dinitrogen by Bacteria and Archaea.

N2O is a potent greenhouse gas and stratospheric reactant that has been steadily on the rise since the beginning of industrialization. It is an obligatory inorganic metabolite of denitrifying bacteria, and some production of N2O is also found in nitrifying and methanotrophic bacteria. We focus this review on the respiratory aspect of N2O transformation catalysed by the multicopper enzyme nitrous oxide reductase (N2OR) that provides the bacterial cell with an electron sink for anaerobic growth. Two types of Cu centres discovered in N2OR were both novel structures among the Cu proteins: the mixed-valent dinuclear Cu(A) species at the electron entry site of the enzyme, and the tetranuclear Cu(Z) centre as the first catalytically active Cu-sulfur complex known. Several accessory proteins function as Cu chaperone and ABC transporter systems for the biogenesis of the catalytic centre. We describe here the paradigm of Z-type N2OR, whose characteristics have been studied in most detail in the genera Pseudomonas and Paracoccus. Sequenced bacterial genomes now provide an invaluable additional source of information. New strains harbouring nos genes and capability of N2O utilization are being uncovered. This reveals previously unknown relationships and allows pattern recognition and predictions. The core nos genes, nosZDFYL, share a common phylogeny. Most principal taxonomic lineages follow the same biochemical and genetic pattern and share the Z-type enzyme. A modified N2OR is found in Wolinella succinogenes, and circumstantial evidence also indicates for certain Archaea another type of N2OR. The current picture supports the view of evolution of N2O respiration prior to the separation of the domains Bacteria and Archaea. Lateral nos gene transfer from an epsilon-proteobacterium as donor is suggested for Magnetospirillum magnetotacticum and Dechloromonas aromatica. In a few cases, nos gene clusters are plasmid borne. Inorganic N2O metabolism is associated with a diversity of physiological traits and biochemically challenging metabolic modes or habitats, including halorespiration, diazotrophy, symbiosis, pathogenicity, psychrophily, thermophily, extreme halophily and the marine habitat down to the greatest depth. Components for N2O respiration cover topologically the periplasm and the inner and outer membranes. The Sec and Tat translocons share the task of exporting Nos components to their functional sites. Electron donation to N2OR follows pathways with modifications depending on the host organism. A short chronology of the field is also presented.

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF‐hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X‐ray structure of human Ca2+‐loaded S100B was determined at 1.9 Å resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size‐exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V‐domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

The cupric site in nitrous oxide reductase contains a mixed‐valence [Cu(II),Cu(I)] binuclear center: A multifrequency electron paramagnetic resonance investigation

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S‐band spectra at 20 K are better resolved than the X‐band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed‐valence [Cu(1.5)..Cu(1.5)] S= 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S‐band spectra to the spectra for the EPR‐detectable Cu of cytochrome c oxidase suggests that the S‐band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.

Cytochrome c nitrite reductase from Wolinella succinogenes. Structure at 1.6 A resolution, inhibitor binding, and heme-packing motifs.

Cytochrome c nitrite reductase catalyzes the 6-electron reduction of nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the ε-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 Å. It is a pentahemec-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes nitrite reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome cnitrite reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochromec nitrite reductases constitute a protein family of high structural similarity.

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes.

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N‐terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane‐bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C‐terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram‐positive bacteria, cyanobacteria and chloroplasts. The residual N‐terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin‐like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (ΔnrfJ,ΔnrfIJ and ΔnrfAIJ) were studied. Mutants ΔnrfAIJ and ΔnrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant ΔnrfJ showed wild‐type properties. The NrfA protein formed by mutant ΔnrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

Multifrequency EPR evidence for a bimetallic center at the CuA site in cytochrome c oxidase.

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in bovine heart cytochrome c oxidase (COX) and nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the existence of Cu‐Cu interaction in both enzymes. C‐band (4.5 GHz) proves to be a particularly good frequency complementing the spectra of COX and N2OR recorded at 2.4 and 3.5 GHz. Both the high and low field region of the EPR spectra show the presence of a well‐resolved 7‐line pattern consistent with the idea of a binuclear Cu center in COX and N2OR. Based on this assumption consistent g‐values are calculated for gz and gx at four frequencies. No consistent g‐values are obtained with the assumption of a 4‐line pattern indicative for a mononuclear Cu site.

A nitrite sensor based on a highly sensitive nitrite reductase mediator-coupled amperometric detection.

Highly sensitive nitrite sensors have been developed for the first time based on mediator-modified electrodes. Tetraheme cytochrome c nitrite reductase from Sulfurospirillum deleyianum and cytochrome cd(1) nitrite reductase from Paracoccus denitrificans are able to accept electrons from artificial electron donors, which simultaneously act as electron mediators between the enzyme and an amperometric electrode. In addition to methyl viologen, redox-active compounds such as phenazines (phenosafranin, safranin T, N-methylphenazinium, 1-methoxy-N-methylphenazinium) and triarylmethane redox dyes (bromphenol blue and red) were selected from a range of redox compounds exhibiting the most efficient performance for nitrite detection. After precipitation, the electron mediators were incorporated in a graphite electrode material. Enzyme immobilization is performed by entrapment in a poly(carbamoyl sulfonate) (PCS) hydrogel. Diffusion coefficients and apparent heterogeneous rate constants of the mediators as well as homogeneous rate constants of nitrite sensors were determined by chronoamperometry and cyclic voltammetry. The phenosafranin-modified electrode layered with the PCS hydrogel immobilization of tetraheme cytochrome c nitrite reductase yielded linear current responses up to 250 μM nitrite with a sensitivity of 446.5 mA M(-)(1) cm(-)(2). The detection limit of the enzymatic nitrite sensor was found to be 1 μM nitrite.