Oliver Einsle

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.

The unprecedented nos gene cluster of Wolinella succinogenes encodes a novel respiratory electron transfer pathway to cytochrome c nitrous oxide reductase

The ε‐proteobacterium Wolinella succinogenes grows anaerobically by respiratory nitrite ammonification but not by denitrification. Nevertheless, it is capable of N2O reduction to N2. Recently, the genome sequence of W. succinogenes revealed a nos gene cluster with intriguing features encoding a new type of N2O reductase. The predicted enzyme is similar to other N2O reductases exhibiting conservation of all residues ligating the two multinuclear copper centers but carries an unprecedented C‐terminal monoheme cytochrome c domain. Notably, the N2O reductase pre‐protein is synthesized with a Sec‐dependent signal peptide, rather than the usually observed twin‐arginine signal sequence, implying that the copper and heme cofactors are both incorporated in the periplasm. The nos gene cluster further consists of four adjacent open reading frames which are predicted to encode two monoheme c‐type cytochromes as well as homologs of NapG and NapH. The latter proteins are thought to function in quinol oxidation coupled to cytochrome c reduction in electron transport to periplasmic nitrate reductase. While the accessory genes nosD, ‐F, ‐Y and ‐L are present in W. succinogenes, homologs of nosR and nosX are absent from the genome. We hypothesize that the nos gene cluster of W. succinogenes encodes a complete electron transport chain catalyzing N2O reduction by menaquinol, a pathway which might also be relevant to other bacteria.

The Amt/Mep/Rh family of ammonium transport proteins (Review)

The Amt/Mep/Rh family of integral membrane proteins comprises ammonium transporters of bacteria, archaea and eukarya, as well as the Rhesus proteins found in animals. They play a central role in the uptake of reduced nitrogen for biosynthetic purposes, in energy metabolism, or in renal excretion. Recent structural information on two prokaryotic Amt proteins has significantly contributed to our understanding of this class, but basic questions concerning the transport mechanism and the nature of the transported substrate, NH3 or , remain to be answered. Here we review functional and structural studies on Amt proteins and discuss the bioenergetic issues raised by the various mechanistic proposals present in the literature.

Helix swapping leads to dimerization of the N-terminal domain of the c-type cytochrome maturation protein CcmH from Escherichia coli

In the process of cytochrome c maturation, heme groups are covalently attached to reduced cysteines of specific heme‐binding motifs (CXXCH) in an apocytochrome c sequence. In Escherichia coli, the CcmH protein maintains apo‐protein cysteines in a reduced state prior to heme attachment. We have purified and biophysically, as well as structurally characterized the soluble, N‐terminal domain of E. coli CcmH that carries the functionally relevant LRCXXC‐motif. In contrast to a recently presented structure of the homologous domain from Pseudomonas aeruginosa, the E. coli protein forms a tightly interlinked dimer by swapping its N‐terminal helix between two monomers. We propose that an altered environment of the functional motif may help to discern between the two redox partners CcmG and apocytochrome c.

Variants of the tetrahaem cytochrome c quinol dehydrogenase NrfH characterize the menaquinol-binding site, the haem c-binding motifs and the transmembrane segment

Members of the NapC/NrfH family are multihaem c-type cytochromes that exchange electrons with oxidoreductases situated at the outside of the cytoplasmic membrane or in the periplasmic space of many proteobacteria. They form a group of membrane-bound quinol dehydrogenases that are essential components of several electron transport chains, for example those of periplasmic nitrate respiration and respiratory nitrite ammonification. Knowledge of the structure-function relationships of NapC/NrfH proteins is scarce and only one high-resolution structure (Desulfovibrio vulgaris NrfH) is available. In the present study, several Wolinella succinogenes mutants that produce variants of NrfH, the membrane anchor of the cytochrome c nitrite reductase complex, were constructed and characterized in order to improve the understanding of the putative menaquinol-binding site, the maturation and function of the four covalently bound haem c groups and the importance of the N-terminal transmembrane segment. Based on amino acid sequence alignments, a homology model for W. succinogenes NrfH was constructed that underlines the overall conservation of tertiary structure in spite of a low sequence homology. The results support the proposed architecture of the menaquinol-binding site in D. vulgaris NrfH, demonstrate that each histidine residue arranged in one of the four CX(2)CH haem c-binding motifs is essential for NrfH maturation in W. succinogenes, and indicate a limited flexibility towards the length and structure of the transmembrane region.

Expression, purification and crystallization of the ammonium transporter Amt-1 from Archaeoglobus fulgidus.

Ammonium transporters (Amts) are a class of membrane-integral transport proteins found in organisms from all kingdoms of life. Their key function is the transport of nitrogen in its reduced bioavailable form, ammonia, across cellular membranes, a crucial step in nitrogen assimilation for biosynthetic purposes. The genome of the hyperthermophilic archaeon Archaeoglobus fulgidus has been annotated with three individual genes for ammonium transporters, amt1-3, the roles of which are as yet unknown. The amt1 gene product has been produced by heterologous overexpression in Escherichia coli and the resulting protein has been purified to electrophoretic homogeneity. Crystals of Amt-1 have been obtained by sitting-drop vapour diffusion and diffraction data have been collected.

Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus

Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron-sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of Ti(III) citrate or dithionite as reducing agents.

Crystal packing of the c6-type cytochrome OmcF from Geobacter sulfurreducens is mediated by an N-terminal Strep-tag II

The putative outer membrane c-type cytochrome OmcF from Geobacter sulfurreducens contains a single haem group and shows homology to soluble cytochromes c(6), a class of electron-transfer proteins that are typically found in cyanobacterial photosynthetic electron-transfer chains. OmcF was overexpressed heterologously in Escherichia coli as an N-terminal Strep-tag II fusion protein and isolated using streptactin-affinity chromatography followed by size-exclusion chromatography. The structure was solved by Fe SAD using data collected to a resolution of 1.86 A on a rotating copper-anode X-ray generator. In the crystal, packing interactions in one dimension were exclusively mediated through the Strep-tag II sequence. The tag and linker regions were in contact with three further monomers of OmcF, leading to a well defined electron-density map for this engineered and secondary-structure-free region of the molecule.

Crystallization of the extracellular rubber oxygenase RoxA from Xanthomonas sp. strain 35Y.

Rubber oxygenase A (RoxA) from Xanthomonas sp. strain 35Y is an extracellular dioxygenase that is capable of cleaving the double bonds of poly(cis-1,4-isoprene) into short-chain isoprene units with 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD) as the major cleavage product. Crystals of the dihaem c-type cytochrome RoxA were grown by sitting-drop vapour diffusion using polyethylene glycol as a precipitant. RoxA crystallized in space group P2(1), with unit-cell parameters a = 72.4, b = 97.1, c = 101.1 A, beta = 98.39 degrees, resulting in two monomers per asymmetric unit. Diffraction data were collected to a limiting resolution of 1.8 A. Despite a protein weight of 74.1 kDa and only two iron sites per monomer, phasing was successfully carried out by multiple-wavelength anomalous dispersion.

Pseudo-merohedral twinning in crystals of the dihaem c-type cytochrome DHC2 from Geobacter sulfurreducens

The dihaem cytochrome DHC2 from Geobacter sulfurreducens has been described as a model system for the study of one of the conserved haem-packing motifs typically observed in multihaem cytochromes c, in which the covalent attachment of haem to the protein allows effective spatial clustering of redox centres and a high haem:protein ratio. Heterologously expressed DHC2 has been crystallized previously and the structure has been solved by multiple-wavelength anomalous dispersion (MAD) and refined to a resolution of 1.5 A (PDB code 2czs). Crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 39.65, b = 55.68, c = 39.64 A, beta = 105.91 degrees . Close inspection of the data indicated the presence of pseudo-merohedral twinning; while not hindering structure solution by MAD, this resulted in high refinement R factors. A re-assessment of the diffraction data for DHC2 is presented, together with a refined structure that includes the twinning present with a twinning fraction alpha of 0.32.