An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase
Identifieur interne : 000861 ( Pmc/Curation ); précédent : 000860; suivant : 000862An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase
Auteurs : Emily Golden [Australie] ; Li-Juan Yu [Australie] ; Flora Meilleur [États-Unis] ; Matthew P. Blakeley [France] ; Anthony P. Duff [Australie] ; Amir Karton [Australie] ; Alice Vrielink [Australie]Source :
- Scientific Reports [ 2045-2322 ] ; 2017.
Abstract
The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.
Url:
DOI: 10.1038/srep40517
PubMed: 28098177
PubMed Central: 5241826
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Blakeley</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Institut Laue-Langevin, 71 Avenue des Martyrs</institution>, Grenoble, 38000,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Duff, Anthony P" sort="Duff, Anthony P" uniqKey="Duff A" first="Anthony P." last="Duff">Anthony P. Duff</name><affiliation wicri:level="1"><nlm:aff id="a5"><institution>Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights NSW</institution>, 2234,<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Karton, Amir" sort="Karton, Amir" uniqKey="Karton A" first="Amir" last="Karton">Amir Karton</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>School of Chemistry and Biochemistry, University of Western Australia</institution>, Crawley, Western Australia, 6009<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Vrielink, Alice" sort="Vrielink, Alice" uniqKey="Vrielink A" first="Alice" last="Vrielink">Alice Vrielink</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>School of Chemistry and Biochemistry, University of Western Australia</institution>, Crawley, Western Australia, 6009<country>Australia</country></nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Becvar, J E" uniqKey="Becvar J">J. E. Becvar</name></author><author><name sortKey="Hastings, J W" uniqKey="Hastings J">J. W. Hastings</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuhlbrandt, W" uniqKey="Kuhlbrandt W">W. Kuhlbrandt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Weber, S" uniqKey="Weber S">S. Weber</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Natarajan, S K" uniqKey="Natarajan S">S. K. Natarajan</name></author><author><name sortKey="Becker, D F" uniqKey="Becker D">D. F. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28098177</article-id><article-id pub-id-type="pmc">5241826</article-id><article-id pub-id-type="pii">srep40517</article-id><article-id pub-id-type="doi">10.1038/srep40517</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Golden</surname><given-names>Emily</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Li-Juan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Meilleur</surname><given-names>Flora</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Blakeley</surname><given-names>Matthew P.</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Duff</surname><given-names>Anthony P.</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Karton</surname><given-names>Amir</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Vrielink</surname><given-names>Alice</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>School of Chemistry and Biochemistry, University of Western Australia</institution>, Crawley, Western Australia, 6009<country>Australia</country></aff><aff id="a2"><label>2</label><institution>Neutron Sciences Directorate, Oak Ridge National Laboratory</institution>, Oak Ridge, TN, 37831,<country>United States of America</country></aff><aff id="a3"><label>3</label><institution>Structural and Molecular Biochemistry, North Carolina State University</institution>, Raleigh, NC 27695,<country>United States of America</country></aff><aff id="a4"><label>4</label><institution>Institut Laue-Langevin, 71 Avenue des Martyrs</institution>, Grenoble, 38000,<country>France</country></aff><aff id="a5"><label>5</label><institution>Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights NSW</institution>, 2234,<country>Australia</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>alice.vrielink@uwa.edu.au</email></corresp></author-notes><pub-date pub-type="epub"><day>18</day><month>01</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>7</volume><elocation-id>40517</elocation-id><history><date date-type="received"><day>27</day><month>09</month><year>2016</year></date><date date-type="accepted"><day>06</day><month>12</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2017, The Author(s)</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>The oxidation reaction catalysed by COx.</title><p>COx catalyses the oxidation of cholesterol to cholest-5-en-3-one <italic>via</italic> a hydride transfer to N5 of FAD and concomitant reduction of the cofactor. A general base abstracts the substrate hydroxyl proton activating the C3-H bond for hydride transfer to FAD.</p></caption><graphic xlink:href="srep40517-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Nuclear density maps for the region between Gly120 and FAD.</title><p><bold>(a)</bold> Nuclear density maps calculated with Gly120-D removed. <bold>(b)</bold> A histogram of selected main chain N-Peak distances. The bin for the Gly120-D peak is indicated by the red arrow. Histogram bin labels refer to the upper limit of the bin and include all distances above the distance value from the previous bin. For example bin label 0.75 represents the distance values 1.65 < x ≤ 1.75 Å. <bold>(c)</bold> Nuclear density maps with a deuteron modelled midway between the Gly120-N and FAD-N5 atoms. <bold>(d)</bold> Nuclear density maps with a deuterium atom modelled at 0.5 occupancy for Gly120-D and 0.5 occupancy on the FAD-N5. Nuclear density (<italic>2F</italic><sub><italic>o</italic></sub> − <italic>F</italic><sub><italic>c</italic></sub>) maps are shown as magenta mesh (1.5 σ) in <bold>(a</bold>,<bold>c)</bold> and <bold>(d)</bold> Residual difference density maps (<italic>F</italic><sub><italic>o</italic></sub> − <italic>F</italic><sub><italic>c</italic></sub>) maps are shown as cyan mesh contoured at 1.5 σ for <bold>(c)</bold> and <bold>(d)</bold> and 3.0 σ for <bold>(a).</bold> Residual density is only shown for a 1.6 Å sphere around the deuteron position shown in <bold>(c)</bold> and <bold>(d)</bold> for clarity. Carbon atoms are colored green, nitrogen atoms are blue, oxygen atoms are red, hydrogen atoms are white and deuterium atoms are yellow.</p></caption><graphic xlink:href="srep40517-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Conservation of a charged residue in the position of Lys225.</title><p><bold>(a)</bold> Nuclear scattering density around Lys225 side chain and secondary interactions with FAD. The positively charged side chain interacts with the main chain O atom of Gly120. <bold>(b)</bold> Superpositions of other oxidoreductases displaying a positively charged residue interacting with the protein carbonyl corresponding to Asn119-O in COx. The N5 atoms of the FAD for each superposed structure are shown as blue spheres (cyan sphere for COx) and the rest of the FAD molecule has been omitted for clarity. Only the C, O and N atoms of the protein main chain are displayed (corresponding to Gly120-O, Gly120-C and Asn119-O). Carbon atoms are colored green, nitrogen atoms are blue, oxygen atoms are red and deuterium atoms are pale yellow. In <bold>(b)</bold>, the COx structure is colored as magenta carbons and cyan nitrogen atoms and the deuterium atoms of Lys225 are colored yellow. The hydrogen atoms interacting with O of the superimposed structures have been colored orange.</p></caption><graphic xlink:href="srep40517-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>A schematic representation of the model system used for obtaining insights into the effects of hydrogen-bond interactions at O1 on the N1–H and N1H•••N5 distances.</title><p><bold>(a)</bold> The peptide bond between Asn119 and Gly120 residue is modeled by <italic>N</italic>-methylformamid, the N5 center of the FAD cofactor is modeled by a substituted pyrazine ring, and hydrogen-bond interactions at O1 of the Asn119 residue are modeled by X<sup>+</sup> = NH<sub>4</sub><sup>+</sup>, H<sub>3</sub>O<sup>+</sup>, and H<sup>+</sup>. <bold>(b)</bold> The hydrogen bond acceptors to Lys225 are provided by the side chains of Leu117 and Asn221 are modeled by water molecules.</p></caption><graphic xlink:href="srep40517-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Orientation of conserved Wat541 and substrate positioning.</title><p><bold>(a)</bold> The nuclear density around conserved Wat541. <bold>(b)</bold> A superposition of dehydroepiandrosterone (DHA) onto the model and density shown in <bold>(a). (c)</bold> A superposition of the DHA model in <bold>(b)</bold> onto the model and residual density of reduced COx presented in Golden, <italic>et al</italic>.<xref ref-type="bibr" rid="b14">14</xref>. Nuclear density (<italic>2F</italic><sub><italic>o</italic></sub> − <italic>F</italic><sub><italic>c</italic></sub>) is displayed as magenta mesh contoured at 1.5 σ. Difference density (<italic>F</italic><sub><italic>o</italic></sub> − <italic>F</italic><sub><italic>c</italic></sub>) is displayed as cyan mesh and contoured at 3.0 σ. Electron density (blue mesh and contoured at 2.0 σ) is displayed only for Wat541 to indicate that its position is localized. Electron difference density is displayed as green mesh (3.0 σ) for the hydride near FAD-N5 in <bold>(c).</bold> Atoms are colored as following: carbon is green, oxygen is red, nitrogen is blue, hydrogen is white, deuterium is yellow except for the DHA model for which carbon atoms are colored magenta and the hydride which is transferred during oxidation is colored cyan.</p></caption><graphic xlink:href="srep40517-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>Model of the interactions which prime the active site for efficient hydride transfer.</title><p>The side chain of Lys225 is stabilised by three hydrogen bonds to carbonyl oxygen atoms (main chain O of Leu117 and Asn119 and side chain of Asn221). Only the A ring of the 3β-hydroxysteroid is shown for clarity. The hydride which is transferred from the substrate to FAD during the oxidation reaction is indicated by the arrow. Atoms involved in localizing the lone pair of electrons on FAD-N5 are highlighted in red.</p></caption><graphic xlink:href="srep40517-f6"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Neutron and X-ray Data Reduction and Refinement Statistics.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50">Neutron</th><th align="center" valign="top" charoff="50">X-ray</th></tr></thead><tbody valign="top"><tr><td colspan="3" align="left" valign="top" charoff="50"><italic><bold>Data Reduction</bold></italic></td></tr><tr><td align="left" valign="top" charoff="50">Resolution (Å)</td><td align="center" valign="top" charoff="50">61.2–2.2 (2.32–2.2)<xref ref-type="fn" rid="t1-fn1">1</xref></td><td align="center" valign="top" charoff="50">40–1.5 (1.58–1.5)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>merge</sub></td><td align="center" valign="top" charoff="50">0.258 (0.349)</td><td align="center" valign="top" charoff="50">0.043 (0.237)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub><italic>pim</italic></sub></td><td align="center" valign="top" charoff="50">0.125 (0.189)</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Observations</td><td align="center" valign="top" charoff="50">70194 (6681)</td><td align="center" valign="top" charoff="50">246246 (32786)</td></tr><tr><td align="left" valign="top" charoff="50">Number unique</td><td align="center" valign="top" charoff="50">17714 (2048)</td><td align="center" valign="top" charoff="50">67723 (9201)</td></tr><tr><td align="left" valign="top" charoff="50">Mean((I)/sd(I))</td><td align="center" valign="top" charoff="50">4 (2.6)</td><td align="center" valign="top" charoff="50">21.1 (4.23)</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">76.7 (61.1)</td><td align="center" valign="top" charoff="50">88.3 (86.9)</td></tr><tr><td align="left" valign="top" charoff="50">Multiplicity</td><td align="center" valign="top" charoff="50">4 (3.3)</td><td align="center" valign="top" charoff="50">3.6 (3.6)</td></tr><tr><td colspan="3" align="left" valign="top" charoff="50"><italic><bold>Refinement</bold></italic></td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>cryst</sub></td><td align="center" valign="top" charoff="50">23.93</td><td align="center" valign="top" charoff="50">13.46</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">28.84</td><td align="center" valign="top" charoff="50">16.87</td></tr><tr><td align="left" valign="top" charoff="50">RMSD bonds</td><td align="center" valign="top" charoff="50">0.014</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">RMSD angles</td><td align="center" valign="top" charoff="50">1.61</td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">Average B</td><td align="center" valign="top" charoff="50">17.3</td><td align="center" valign="top" charoff="50"> </td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p><sup>1</sup>Statistics for highest resolution bin are shown in parentheses.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t2"><label>Table 2</label><caption><title>Effects of Hydrogen-Bonds at O1 on the N1–H and N1H•••N5 Bond Distances and Atomic Charge on N1 of the Gly120 Residue Calculated at the B2PLYP-D3BJ/Def2-TZVPP Level of Theory.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="center" valign="top" charoff="50">H-bond donor</th><th colspan="2" align="center" valign="top" charoff="50">Δ N1–H bond distance<xref ref-type="fn" rid="t2-fn1">2</xref></th><th align="center" valign="top" charoff="50">Δ N1H•••N5 distance<xref ref-type="fn" rid="t2-fn2">3</xref></th><th align="center" valign="top" charoff="50">Charge on N1<xref ref-type="fn" rid="t2-fn5">6</xref></th></tr><tr><th align="center" valign="top" charoff="50">Without FAD</th><th align="center" valign="top" charoff="50">With FAD</th><th align="center" valign="top" charoff="50">With FAD</th><th align="center" valign="top" charoff="50">Without FAD</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">None</td><td align="center" valign="top" charoff="50">0.000<xref ref-type="fn" rid="t2-fn3">4</xref></td><td align="center" valign="top" charoff="50">0.010</td><td align="center" valign="top" charoff="50">0.000<xref ref-type="fn" rid="t2-fn4">5</xref></td><td align="center" valign="top" charoff="50">−0.664</td></tr><tr><td align="left" valign="top" charoff="50">NH<sub>4</sub><sup>+</sup></td><td align="center" valign="top" charoff="50">0.003</td><td align="center" valign="top" charoff="50">0.046</td><td align="center" valign="top" charoff="50">−0.267</td><td align="center" valign="top" charoff="50">−0.633</td></tr><tr><td align="left" valign="top" charoff="50">H<sub>3</sub>O<sup>+</sup></td><td align="center" valign="top" charoff="50">0.006</td><td align="center" valign="top" charoff="50">0.055</td><td align="center" valign="top" charoff="50">−0.308</td><td align="center" valign="top" charoff="50">−0.526</td></tr><tr><td align="left" valign="top" charoff="50">H<sup>+</sup></td><td align="center" valign="top" charoff="50">0.008</td><td align="center" valign="top" charoff="50">0.077</td><td align="center" valign="top" charoff="50">−0.384</td><td align="center" valign="top" charoff="50">−0.464</td></tr><tr><td align="left" valign="top" charoff="50">NH<sub>4</sub><sup>+</sup> <xref ref-type="fn" rid="t2-fn6">7</xref></td><td align="center" valign="top" charoff="50">0.001</td><td align="center" valign="top" charoff="50">0.025</td><td align="center" valign="top" charoff="50">−0.139</td><td align="center" valign="top" charoff="50">−0.664</td></tr><tr><td align="left" valign="top" charoff="50">H<sub>3</sub>O<sup>+</sup> <xref ref-type="fn" rid="t2-fn7">8</xref></td><td align="center" valign="top" charoff="50">0.003</td><td align="center" valign="top" charoff="50">0.041</td><td align="center" valign="top" charoff="50">−0.245</td><td align="center" valign="top" charoff="50">−0.626</td></tr></tbody></table><table-wrap-foot><fn id="t2-fn1"><p><sup>2</sup>The tabulated numbers are the increase in the N1–H bond length (in Å) relative to that in free N-methylformamid, the model system used for modeling the H-bond-donor•••O1 = C–N1–H•••FAD complex is shown in <xref ref-type="fig" rid="f4">Fig. 4</xref>.</p></fn><fn id="t2-fn2"><p><sup>3</sup>The tabulated numbers are the decreases in the N1H•••N5 bond length (in Å) relative to that in free N-methylformamid (see <xref ref-type="fig" rid="f4">Fig. 4</xref>).</p></fn><fn id="t2-fn3"><p><sup>4</sup>Reference distance (N1–H) = 1.004 Å.</p></fn><fn id="t2-fn4"><p><sup>5</sup>Reference distance (N1H•••N5) = 2.014 Å.</p></fn><fn id="t2-fn5"><p><sup>6</sup>Atomic polar tensor (APT) charge on N1 in atomic units.</p></fn><fn id="t2-fn6"><p><sup>7</sup>Added two water molecules to form hydrogen bond with NH<sub>4</sub><sup>+</sup> (see <xref ref-type="fig" rid="f4">Fig. 4b</xref>).</p></fn><fn id="t2-fn7"><p><sup>8</sup>Added two water molecules to form hydrogen bond with H<sub>3</sub>O<sup>+</sup>.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document 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