Active sites without restraints: high-resolution analysis of metal cofactors

Burger, Eva-Maria; Andrade, Susana L. A.; Einsle, Oliver

doi:10.1016/j.sbi.2015.07.016

Cited by 9 publications

(11 citation statements)

References 38 publications

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“…Because of the relatively small X‐ray absorption cross‐section, one usually needs relatively high concentrations to obtain detectable signals, of the order of a few millimolar. Moreover, the intense incident X‐ray beam often causes sample changes and/or degradation, e.g., photoreduction or lysis,, which can be avoided by limiting the time the sample is exposed to the beam and repeating the individual scans several times at fresh sample spots. Sensitive biological samples often survive only tens of seconds of exposure at typical modern synchrotron beams before spectral changes are observed, even at 10 K or lower ,.…”

Section: Instrumentationmentioning

confidence: 99%

A Practical Guide to High‐resolution X‐ray Spectroscopic Measurements and their Applications in Bioinorganic Chemistry

Kowalska

Lima

Pollock

et al. 2016

Israel Journal of Chemistry

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The bioinorganic chemistry community has had a long history of utilizing a diverse range of spectroscopic techniques to obtain detailed geometric and electronic structural insights into metalloprotein active sites. In recent years, the development of beamlines optimized for high‐resolution X‐ray spectroscopy has provided novel tools for the elucidation of biological active site structures. These methods include both non‐resonant and resonant X‐ray emission spectroscopy. Herein, we briefly introduce both X‐ray absorption and X‐ray emission spectroscopy, and the added information that can be obtained through high‐resolution measurements. The information content of each spectroscopic approach, as well as the required instrumentation, is discussed. Applications of these methods in bioinorganic chemistry are highlighted.

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Section: Instrumentationmentioning

confidence: 99%

A Practical Guide to High‐resolution X‐ray Spectroscopic Measurements and their Applications in Bioinorganic Chemistry

Kowalska

Lima

Pollock

et al. 2016

Israel Journal of Chemistry

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“…The W-O distance of 2.04 Å observed in the X-ray structure at 1.26 Å resolution was between the values expected for an hydroxo ligand (OH − , 1.9-2.1 Å) and a coordinated water molecule (H 2 O, 2.0-2.3 Å). The authors chose H 2 O, since the close proximity of the heavy scatterer W may distort the distance observed in the X-ray data by Fourier series termination and a simulation of this effect resulted in a true ligand distance of 2.25 Å [63,70].…”

Section: Active Site Cavity Of Ah and Site-directed Mutagenesismentioning

confidence: 99%

“…Problems concerning high quality crystal structures of metal-and cofactor-containing proteins, particularly when light ligands are bound to or are in close proximity of heavy atoms, have been recently pointed out by Einsle and colleagues [70]. A coordinated H 2 O could gain a partially positive net charge by the proximity of the protonated Asp-13, turning it into an electrophile that would directly attack the C≡C triple bond.…”

Section: Mechanism Of Ahmentioning

confidence: 99%

Acetylene hydratase: a non-redox enzyme with tungsten and iron–sulfur centers at the active site

Kroneck

2016

J Biol Inorg Chem

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In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe-4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C2H2. To access the W-Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C2H2 binds directly to W. The architecture of the catalytic cavity, the specificity for C2H2 and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C2H2 are needed to present a conclusive answer.

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“…The original 2.2 Å resolution structure of nitrogenase detailed an FeMoco with a geometrically complex arrangement of six inner iron atoms and nine surface sulfur atoms . This arrangement of identical scatterers resulted in additive ripple effects from each atom, effectively concealing the central light atom and leading to the initial incorrect interpretation of an empty pocket . In a later 1.16 Å resolution structure, a light atom at the center of the FeMoco was observed but unable to be unambiguously assigned .…”

Section: Introductionmentioning

confidence: 96%

Metalloprotein Crystallography: More than a Structure

2016

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ConspectusMetal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25–50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV–vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.

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Active sites without restraints: high-resolution analysis of metal cofactors

Cited by 9 publications

References 38 publications

A Practical Guide to High‐resolution X‐ray Spectroscopic Measurements and their Applications in Bioinorganic Chemistry

A Practical Guide to High‐resolution X‐ray Spectroscopic Measurements and their Applications in Bioinorganic Chemistry

Acetylene hydratase: a non-redox enzyme with tungsten and iron–sulfur centers at the active site

Metalloprotein Crystallography: More than a Structure

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