H/D exchange rates of the secondary structure elements.

<p>**P<0.001 in comparison to the value for rArom.</p><p>*P<0.005 in comparison to the value for rArom.</p

Access channel of rArom for the substrate androstenedione.

<p>A) Overall structure of rArom (PDB ID 4KQ8). The most flexible regions according to the MD simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082118#pone.0082118-Jiang1" target="_blank">[18]</a> are shown in red. The parts of the secondary structure elements forming the access channel are shown in yellow (helix E), cyan (helix F), orange (helix)I, magenta (β-strand 9). B) Zoom in the active site: the heme is shown in red, the Van der Waals surface of androstenedione (blue) and Trp224 (grey) are also shown.</p

FTIR spectra of rArom during H/D exchange.

<p>A) Spectra recorded as a function of time after connecting the sample cell with a ²H₂O-saturated nitrogen flow. The spectra are normalized for the amide I signal. B) Second derivative spectra obtained from the FTIR spectra recorded as a function of time. The signals at 1655, 1623 and 1640 cm⁻¹ are assigned to α-helices, β-sheets and random coil, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082118#pone.0082118-Jung2" target="_blank">[49]</a>.</p

Fluorescence properties of rArom and the mutant Trp224Phe.

<p>A) Steady-state fluorescence emission spectra (λ_exc = 293 nm) of rArom in the absence (solid line) and in the presence of the substrate androstenedione (dashed line) or the inhibitor anastrozole (dotted line). B) Phase-shift and demodulation data of rArom in the absence (squares), and in the presence of the substrate androstenedione (circles) or the inhibitor anastrozole (triangles). C) Fluorescence lifetimes obtained from the fitting of the experimental data to a four discrete components model for rArom in the absence (black bars), and in the presence of the substrate androstenedione (dashed bars) or the inhibitor anastrozole (dotted bars). Panels D), E) and F) use the same symbols to show the results obtained under the same conditions for mutant Trp224Phe.</p

Amount of secondary structure elements (%) present in rArom and comparison with the values reported in literature for other P450 enzymes.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082118#pone.0082118-Jung2" target="_blank">[49]</a>.^a from ref. </p

Absorption spectroscopy of rArom.

<p>A) Visible absorption spectrum of 3.8 µM rArom in the absence of substrate (thick black line) and in the presence of 10 µM of the substrate androstenedione (thin black line) and 1 µM of the inhibitor anastrozole (grey line). B) Difference spectrum of Fe(II)-CO minus Fe(II) of rArom purified in absence of any substrate/inhibitor. C) Binding curve of androstenedione (inset: spectra recorded during substrate titration). D) Thermal denaturation of rArom in the absence (squares), and in the presence of the substrate androstenedione (circles) or the inhibitor anastrozole (triangles) monitored as a decrease in ellipticity at 208 nm in the far-UV CD spectra. The data were fit to a two-state transition to obtain T_m for rArom in the absence (light grey curve), and presence of the substrate androstenedione (dark grey curve) or the inhibitor anastrozole (black curve).</p