Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles ϕ and ψ (Cornilescu et al. J Biomol NMR 13 289–302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted ϕ and ψ angles, equals ±13°. Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

/pdf/talos-a-hybrid-method-for-predicting-protein-backbone-4t5z8ivciv.pdf

TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.

An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic β subunits that support the binding change mechanism with an internal rotation of the γ subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catalytic site occupancy and behavior during hydrolysis or synthesis of ATP. Subunit function as related to proton translocation and rotational catalysis is considered. Physical demonstrations of the γ subunit rotation have been achieved. The findings have implications for other enzymatic catalyses.

/pdf/the-atp-synthase-a-splendid-molecular-machine-46l38xgcv7.pdf

The ATP Synthase—A Splendid Molecular Machine

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTHydrogenasesWolfgang Lubitz*, Hideaki Ogata, Olaf Rüdiger, and Edward ReijerseView Author Information Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, 45470 Mülheim an der Ruhr, Germany*E-mail: [email protected]Cite this: Chem. Rev. 2014, 114, 8, 4081–4148Publication Date (Web):March 21, 2014Publication History Received16 October 2013Published online21 March 2014Published inissue 23 April 2014https://pubs.acs.org/doi/10.1021/cr4005814https://doi.org/10.1021/cr4005814review-articleACS PublicationsCopyright © 2014 American Chemical SocietyRequest reuse permissionsArticle Views26977Altmetric-Citations1637LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Cluster chemistry,Ligands,Monomers,Peptides and proteins,Redox reactions Get e-Alerts 

Hydrogenases.

Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/β-mixed or β-structure–rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.

https://www.pnas.org/content/pnas/112/24/E3095.full.pdf

Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy

Interaction of metal ions with phosphatidylcholine bilayer membranes.

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser20-Lys41) of β2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13C,15N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a β-strand–loop–β-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of β2-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.

https://www.pnas.org/content/pnas/103/48/18119.full.pdf

3D structure of amyloid protofilaments of beta2-microglobulin fragment probed by solid-state NMR

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.

/pdf/amyloid-protofilament-formation-of-hen-egg-lysozyme-in-3r02jfkn9a.pdf

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.

We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of ≈10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on 13C dipolar spin-diffusion solid-state NMR of uniformly 13C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular CC distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the ≈90 intermolecular distances that BChl c molecules form piggyback-dimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.

https://www.pnas.org/content/pnas/104/3/790.full.pdf

Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR

For clarification of the assignment of the vibrational modes of the choline group, Raman spectra of choline iodides selectively deuterated at three different positions were investigated. The isotope shifts of the C-N stretching vibrations suggested that they are conformation sensitive. When the Raman spectra of choline chloride, carbamoylcholine iodide, carbamoylcholine chloride, and methoxycarbonylcholine iodide are compared with the crystal structures of these compounds, a correlation between the vibrational frequency and the conformation of the O-C-C-N+ backbone could be established. The Raman bands attributed to the "totally" symmetric stretching (v1) and symmetric stretching vibrations (v2) of the C-N bonds of the quaternary ammonium group appeared at about 720 cm-1 and about 870 cm-1, respectively, for the gauche conformation of the O-C-C-N+ backbone, and in the trans conformation, they shifted to about 770 cm-1 (v1) and about 910 cm-1 (v2), respectively. On the basis of this correlation and from measurements of phosphatidylcholine and sphingomyelin bilayers, it was concluded that most of the choline groups in both bilayers take the gauche conformation not only in the solid state but also in the gel and liquid-crystalline states. These data represent the first direct evidence that a gauche conformation for the O-C-C-N+ bond is preferred in the gel and liquid-crystalline states. These key bands, especially the v1 band, are a powerful tool to study the conformation of the choline group in situ not only in the membrane field but also in the neuroscience in connection with acetylcholine.

Direct determination by Raman scattering of the conformation of the choline group in phospholipid bilayers

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