Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome<i>c</i>nitrite reductase

Trofimov, A.A.; Polyakov, K. M.; Лазаренко, В. А.; Попов, А. Н.; Tikhonova, Т. V.; Tikhonov, A.V.; Попов, В.О.

doi:10.1107/s1399004715003053

Cited by 8 publications

(7 citation statements)

References 36 publications

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“…14,15 The crystallographic studies revealed CcNiR to be a homodimeric multiheme enzyme in all bacterial organisms except in the haloalkaliphilic bacterium T. nitratireducens. 14 The latter exists as a stable hexamer containing 48 hemes.…”

Section: Introductionmentioning

confidence: 99%

Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies

Bykov

Neese

2015

Inorg. Chem.

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In this Forum Article, an extensive discussion of the mechanism of six-electron, seven-proton nitrite reduction by the cytochrome c nitrite reductase enzyme is presented. On the basis of previous studies, the entire mechanism is summarized and a unified picture of the most plausible sequence of elementary steps is presented. According to this scheme, the mechanism can be divided into five functional stages. The first phase of the reaction consists of substrate binding and N-O bond cleavage. Here His277 plays a crucial role as a proton donor. In this step, the N-O bond is cleaved heterolytically through double protonation of the substrate. The second phase of the mechanism consists of two proton-coupled electron-transfer events, leading to an HNO intermediate. The third phase involves the formation of hydroxylamine, where Arg114 provides the necessary proton for the reaction. The second N-O bond is cleaved in the fourth phase of the mechanism, again triggered by proton transfer from His277. The Tyr218 side chain governs the fifth and last phase of the mechanism. It consists of radical transfer and ammonia formation. Thus, this mechanism implies that all conserved active-site side chains work in a concerted way in order to achieve this complex chemical transformation from nitrite to ammonia. The Forum Article also provides a detailed discussion of the density functional theory based cluster model approach to bioinorganic reactivity. A variety of questions are considered: the resting state of enzyme and substrate binding modes, interaction with the metal site and with active-site side chains, electron- and proton-transfer events, substrate dissociation, etc.

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Section: Introductionmentioning

confidence: 99%

Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies

Bykov

Neese

2015

Inorg. Chem.

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“…The inelastic interactions of X- rays with the protein in the crystal during data collection can lead to crystal radiation damage and different chemical modifications of the protein 70 . One specific effect of radiation damage to the protein is the reduction of some metal ions 71–73 . During the crystallographic experiment, it is important to consider and address the possibility of the metal reduction, especially if the metal is expected to be in a higher oxidation state when multiple oxidation states are possible in a protein-metal complex.…”

Section: Introductionmentioning

confidence: 99%

Characterizing metal-binding sites in proteins with X-ray crystallography

et al. 2018

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Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

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“…Inelastic X-ray scattering produces photoelectrons that can change the oxidation state of metals in the active sites of redox enzymes and cause an enzymatic reaction (Garman, 2010). The serial single-crystal method had previously been applied to study enzymatic catalysis in crystals of metal-mediated reductases (Trofimov et al, 2015;Horrell et al, 2016). The connection of the copper-ion oxidation to its coordination at photoreduction has been explored in the case of monooxygenases (Hemsworth et al, 2013;Gudmundsson et al, 2014;Frandsen et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism

Polyakov

Gavryushov

Федорова

et al. 2019

Acta Crystallogr D Struct Biol

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Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X‐ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X‐ray diffraction data sets were collected with increasing doses of absorbed X‐ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccase crystal confirmed the previously deduced enzymatic mechanism of molecular oxygen reduction. The structures of the complexes allowed the localization of chloride and fluoride ions in the channel near the T2 copper ion. These ions replace the oxygen ligand of the T2 copper ion in this channel and can play the role of this ligand in the enzymatic reaction. As follows from analysis of the structures from the increasing dose series, the inhibition of laccases by chloride and fluoride anions can be explained by the fact that the binding of these negatively charged ions at the position of the oxygen ligand of the T2 copper ion impedes the reduction of the T2 copper ion.

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Structural study of the X-ray-induced enzymatic reaction of octahaem cytochromecnitrite reductase

Cited by 8 publications

References 36 publications

Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies

Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies

Characterizing metal-binding sites in proteins with X-ray crystallography

The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism

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