Wolfram Meyer-Klaucke

The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site.

Biological formation and consumption of molecular hydrogen (H2) are catalyzed by hydrogenases, of which three phylogenetically unrelated types are known: [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenase. We present a crystal structure of [Fe]-hydrogenase at 1.75 angstrom resolution, showing a mononuclear iron coordinated by the sulfur of cysteine 176, two carbon monoxide (CO) molecules, and the sp2-hybridized nitrogen of a 2-pyridinol compound with back-bonding properties similar to those of cyanide. The three-dimensional arrangement of the ligands is similar to that of thiolate, CO, and cyanide ligated to the low-spin iron in binuclear [NiFe]- and [FeFe]-hydrogenases, although the enzymes have evolved independently and the CO and cyanide ligands are not found in any other metalloenzyme. The related iron ligation pattern of hydrogenases exemplifies convergent evolution and presumably plays an essential role in H2 activation. This finding may stimulate the ongoing synthesis of catalysts that could substitute for platinum in applications such as fuel cells.

Architecture of a protein central to iron homeostasis : crystal structure and spectroscopic analysis of the ferric uptake regulator.

Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur). Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 Å. Furthermore, X‐ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.

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.

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.

HDAC6–p97/VCP controlled polyubiquitin chain turnover

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin‐binding domain of HDAC6, named ZnF‐UBP, and investigated its biological functions. These studies show that the three Zn ion‐containing HDAC6 ZnF‐UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6‐interacting chaperone, p97/VCP, dissociates the HDAC6–ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.

Identifying the Minimal Copper- and Zinc-binding Site Sequence in Amyloid-β Peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-β (Aβ) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Aβ peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Aβ1-40, Aβ1-16, Aβ1-28, Aβ5-23, and Aβ17-40). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His6, His13, and His14). In zinc-Aβ complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Aβ peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.

Metal binding in amyloid β-peptides shows intra- and inter-peptide coordination modes

X-ray absorption spectroscopy data show different metal binding site structures in β-amyloid peptides according to whether they are complexed with Cu2+ or Zn2+ ions. While the geometry around copper is stably consistent with an intra-peptide binding with three metal-coordinated Histidine residues, the zinc coordination mode depends on specific solution conditions. In particular, different sample preparations are seen to lead to different geometries around the absorber that are compatible with either an intra- or an inter-peptide coordination mode. This result reinforces the hypothesis that assigns different physiological roles to the two metals, with zinc favoring peptide aggregation and, as a consequence, plaque formation.

The iron-sulfur-cluster-free hydrogenase (Hmd) is a metalloenzyme with a novel iron binding motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Crystal Structure and Function of the Zinc Uptake Regulator FurB from Mycobacterium tuberculosis

Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for novel antibiotics. Here we report the crystal structure of FurB from Mycobacterium tuberculosis at a resolution of 2.7Å, and we present biochemical and spectroscopic data that allow us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerization domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis presented here reveals that M. tuberculosis FurB is Zn(II)-dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic, and biochemical results enables us to determine the structural basis for functional differences in this important family of bacterial regulators.

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.