Metalloprotein active site structure determination: Synergy between X-ray absorption spectroscopy and X-ray crystallography

Cotelesage, Julien J. H.; Pushie, M. Jake; Grochulski, P.; Pickering, Ingrid J.; George, Graham N.

doi:10.1016/j.jinorgbio.2012.06.019

Cited by 75 publications

(97 citation statements)

References 83 publications

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“…EXAFS spectra were simulated using both filtered and unfiltered data; however, simulation results are presented only for fits to raw (unfiltered) data. Simulation protocols and criteria for determining the best fit have been described previously (63). …”

Section: Xas Analysismentioning

confidence: 99%

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

Ruetz

Kumutima

Lewis

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellHydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H 2 S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H 2 S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H 2 S tightly and was postulated to play a role in modulating neuronal sensitivity to H 2 S in conditions such as stroke. However, the H 2 S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H 2 S. Ferric neuroglobin is slowly reduced by H 2 S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His 64 residue to alanine promotes rapid binding of H 2 S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H 2 S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2-6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His 64 ligand.Neuroglobin is a member of the globin family (1) that is expressed primarily in neuronal tissues (2) but also in other metabolically highly active endocrine tissues, such as the adrenal gland and the pancreatic islets of Langerhans (3, 4). Although the physiological role of neuroglobin is still elusive, early studies suggested that it might be involved in oxygen supply (1, 5). However, neuroglobin is generally expressed at low levels and exhibits a high autoxidation rate, producing reactive oxygen species, which makes its role as an O 2 carrier unlikely (6). Neuroglobin can also scavenge reactive oxygen species and reduce nitrite to NO under hypoxic conditions (7-9).In contrast to hemoglobin and myoglobin, two histidine residues coordinate the heme cofactor in neuroglobin in the ferrous and ferric states (Fig. 1A). The crystal structure of neuroglobin reveals a high structural similarity to myoglobin, despite Ͻ25% amino acid sequence similarity between the proteins. The structure reveals a large hydrophobic cavity, which connects the proximal and distal sides of the heme (7). His 64 and His 96 coordinate the heme on the distal and proximal sides, respectively (10). Exogenous ligands like O 2 , CO, and NO bind to ferrous neuroglobin (Fe II -Ngb) 2 on the distal side, and binding is limited by dissociation of His 64 , which is slow (6,(11)(12)(13)(14). Limited characterization of the binding of sulfide to ferric neuroglobin (Fe III -Ngb) has...

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Section: Xas Analysismentioning

confidence: 99%

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

Ruetz

Kumutima

Lewis

et al. 2017

Journal of Biological Chemistry

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“…We examined the EXAFS portion of the X-ray absorption spectrum, which can be used with quantitative analysis to obtain a radial structure [27]. EXAFS spectra of methylmercury and inorganic mercury in the presence of PTU, together with the associated Fourier transforms, are shown in Fig.…”

Section: X-ray Fluorescence Imaging (Xfi)mentioning

confidence: 99%

Phenylthiourea alters toxicity of mercury compounds in zebrafish larvae

MacDonald

Nehzati

Sylvain

et al. 2015

Journal of Inorganic Biochemistry

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In recent years larval stage zebrafish have been emerging as a standard vertebrate model in a number of fields, ranging from developmental biology to pharmacology and toxicology. The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is used very widely with larval zebrafish to generate essentially transparent organisms through inhibition of melanogenesis, which has enabled many elegant studies in areas ranging from neurological development to cancer research. Here we show that PTU can have dramatic synergistic and antagonistic effects on the chemical toxicology of different mercury compounds. Our results indicate that extreme caution should be used when employing PTU in toxicological studies, particularly when studying toxic metal ions.

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“…However, because of a limited, but significant mobility of atoms in proteins at the cryogenic temperatures (around 100 K) usually employed in crystallography, XPR-induced and native structures of cofactors in reduced states may be expected to be different (58,60). By x-ray absorption spectroscopy (XAS), structural (metalligand bond lengths, metal-metal distances) and electronic (oxidation state) parameters also for high-valent metal sites can be determined (63)(64)(65) and furthermore, this technique allows studies of the XPR kinetics (58 -60, 66). Using XAS, for CtR2 we have shown previously that under high-intensity x-ray irradiation the Mn(IV)Fe(III) cofactor becomes reduced by XPR within seconds to minutes even at 20 K (30).…”

mentioning

confidence: 99%

“…This is necessary to reconcile the site configurations in the crystal data with deviating structural features, for example, usually much shorter metal-metal distances, as determined by XAS (29, 30, 66 -68). In principle, this would allow for the reversion of modifications due to XPR in the crystal structures in silico, using quantum chemical calculations (64,69,70), to obtain improved models of the high-valent sites.…”

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confidence: 99%

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Sigfridsson

Chernev

Leidel

et al. 2013

Journal of Biological Chemistry

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Background: Typical FeFe and MnFe cofactors bind to numerous enzymes such as ribonucleotide reductases. Crystallographic data suggest x-ray photoreduction (XPR) effects. Results: Rapid XPR-induced cofactor changes were monitored using time-resolved x-ray absorption spectroscopy. Conclusion: The XPR-induced cofactor states differ significantly from the native configurations, but comply with crystallographic structures. Significance: Structure determination for high-valent dimetal-oxygen cofactors requires free electron-laser protein crystallography combined with x-ray spectroscopy.

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Metalloprotein active site structure determination: Synergy between X-ray absorption spectroscopy and X-ray crystallography

Cited by 75 publications

References 83 publications

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

Phenylthiourea alters toxicity of mercury compounds in zebrafish larvae

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

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