13 research outputs found
Experimental Quantification of Electrostatics in X–H···π Hydrogen Bonds
Hydrogen bonds are ubiquitous in chemistry and biology.
The physical
forces that govern hydrogen-bonding interactions have been heavily
debated, with much of the discussion focused on the relative contributions
of electrostatic vs quantum mechanical effects. In principle, the
vibrational Stark effect, the response of a vibrational mode to electric
field, can provide an experimental method for parsing such interactions
into their electrostatic and nonelectrostatic components. In a previous
study we showed that, in the case of relatively weak O–H···π
hydrogen bonds, the O–H bond displays a linear response to
an electric field, and we exploited this response to demonstrate that
the interactions are dominated by electrostatics (Saggu, M.; Levinson,
N. M.; Boxer, S. G. <i>J. Am. Chem. Soc.</i> <b>2011</b>, <i>133</i>, 17414–17419). Here we extend this
work to other X–H···π interactions. We
find that the response of the X–H vibrational probe to electric
field appears to become increasingly nonlinear in the order O–H
< N–H < S–H. The observed effects are consistent
with differences in atomic polarizabilities of the X–H groups.
Nonetheless, we find that the X–H stretching vibrations of
the model compounds indole and thiophenol report quantitatively on
the electric fields they experience when complexed with aromatic hydrogen-bond
acceptors. These measurements can be used to estimate the electrostatic
binding energies of the interactions, which are found to agree closely
with the results of energy calculations. Taken together, these results
highlight that with careful calibration vibrational probes can provide
direct measurements of the electrostatic components of hydrogen bonds
Direct Measurements of Electric Fields in Weak OH···π Hydrogen Bonds
Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···π hydrogen bonds. The effects of complex formation on the vibrational frequency of the phenol OH or OD stretches were measured in a series of benzene-based aromatic solvents. Large shifts are observed and these can be converted into electric fields via the measured vibrational Stark effect. A comparison of the measured fields with quantum chemical calculations demonstrates that calculations performed in the gas phase are surprisingly effective at capturing the electrostatics observed in solution. The results provide quantitative measurements of the magnitude of electric fields and electrostatic binding energies in these interactions and suggest that electrostatics dominate them. The combination of vibrational Stark effect (VSE) measurements of electric fields and high-level quantum chemistry calculations is a general strategy for quantifying and characterizing the origins of intermolecular interactions
EPR linewidths and g-values of FeSI and FeSII from mAOX1.
a<p>AOX wild-type from rabbit liver, values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#pone.0005348-Stesmans1" target="_blank">[36]</a>.</p>b<p>g-strain was included in the simulation with 0.01 for g<sub>z</sub> .</p>c<p>g-strain was included in the simulation with 0.04 for g<sub>x</sub> . Estimated error of g-values: ±0.004 for FeSI and ±0.008 for FeSII.</p
Purification of recombinant mAOX1 after expression in <i>E. coli</i> TP1000 cells.
a<p>Total protein was quantified with the Bradford assay.</p>b<p>The activity was measured by monitoring the decrease in absorption at 600 nm in the presence of 500 µM benzaldehyde and 100 µM DCPIP.</p>c<p>Specific enzyme activity (units/mg) is defined as the oxidation of 1 µM benzaldehyde per min and mg of enzyme under the assay conditions.</p
Characterization of wild-type mAOX1 by UV-VIS absorption spectroscopy.
<p>Spectra of 7 µM of the air-oxidized mAOX1 in 50 mM Tris, 1 mM EDTA, pH 7.5, under anaerobic conditions.</p
Steady-state kinetic parameters of recombinant mAOX1 and variants with different aldehyde and purine substrates.
a<p>Apparent kinetic parameters were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5 by varying the concentration of substrate in the presence of 100 µM DCPIP as electron acceptor.</p><p>n.d., none was detectable.</p><p>-, not determined.</p
EPR spectra of mAOX1 wild-type.
<p>Experimental cw-EPR spectra of dithionite-reduced mAOX1 wild-type samples at pH 7.0 (trace a) together with the corresponding simulation (trace b). For simulation parameters see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005348#pone-0005348-t003" target="_blank">Table 3</a>. The flavin semiquinone was simulated with an isotropic g-value of g<sub>iso</sub> = 2.0 and 1.9 mT (trace e). (MoV) was neglected in all simulations. (a) mAOX1 wild-type; (b) simulation of complete spectrum; (c) simulation of FeSI; (d) simulation of FeSII; (e) simulation of FAD. Experimental conditions: T = 20 K, 1 mW microwave power, 1 mT modulation amplitude, 12.5 kHz modulation frequency.</p
Steady-state kinetic parameters of recombinant <i>R. capsulatus</i> XDH and variants with different aldehyde and purine substrates.
a<p>Apparent kinetic parameters were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5 by varying the concentration of substrate in the presence of 100 µM DCPIP as electron acceptor.</p><p>n.d., none was detectable.</p><p>-, not determined.</p
CD-Spectroscopy of mAOX1 wild-type.
<p>Spectra were recorded in 50 mM Tris, 1 mM EDTA, pH 7.5 at 10°C using a Jasco J-715 CD-spectrometer. Spectra of 2.1 mg/mL of mAOX1 were recorded in the oxidized state (solid lines) and after reduction with sodium dithionite (dotted lines).</p
Native PAGE of mAOX1 wild-type and variants after purification.
<p>Purified enzymes were analyzed by 7% native PAGE. Each lane contained 6 µg of purified enzyme: lane 1, mAOX1 wild-type; lane 2, mAOX1-V806E; lane 3, mAOX1-M884R; lane 4, mAOX1-V806E/M884R; lane 5, mAOX1-E1265Q.</p