Supplementary Material:
Teresa Miletti, † Justin Di Trani, † Louis-Charles Jr. Levros, ‡ Anthony Mittermaier* †
† Department of Chemistry, McGill University, Montreal, QC, H3A 0B8, Canada.
‡Laboratoire de biologie moléculaire, Département des Sciences Biologiques, and Centre
BioMed, Université du Québec à Montréal, Montréal, Québec, H3C 3P8, Canada
Corresponding Author:
Anthony Mittermaier
Department of Chemistry
McGill University
801 Sherbrooke St. W. Room 322
Montreal, QC, Canada
H3A 0B8
Office: (514) 398-3085
Fax: (514) 398-3797 anthony.mittermaier@mcgill.ca

Figure S1. Overlay of NOX NMR spectra obtained from a titration with urea. Spectra were collected with a Varian INOVA spectrometer operating at 800 MHz and 50°C on NOX samples containing 2.4mM
FMN, 50mM potassium phosphate buffer (pH 7.2) and urea at concentrations of (red) 0 M, 0.2M, 0.4M,
0.6M, 0.8M, 1.0M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M urea (blue).

Figure S2. Overlay of 1 H/ 15 N HSQC NMR spectra of NOX obtained over a temperature range of 25 to
50°C. Spectra were collected with a Varian INOVA spectrometer operating at 800 MHz and (blue) 25°C,
30°C, 35°C, 40°C, 45°C, 50°C (red) on NOX samples containing 2.4mM FMN and 50mM potassium phosphate buffer (pH 7.2).

Figure S3. Overlaid 1 H/ 15 N HSQC NMR spectra of NOX obtained from titrations of FMN and FAD. The overlay consists of spectra collected for NOX with (a) FMN concentrations of (red) 0.00, 0.05, 0.15, 0.20,
0.25, 0.30, 0.50, 1.00, 2.00, 2.40, 3.00 and 5.00 mM (blue), (b) FAD concentrations of (red) 0.0, 0.2, 0.4,
1.0, 2.0, 2.5, 3.0, 3.5, 14.9 and 39.9 mM (blue) at 50°C in 50mM potassium phosphate buffer at pH 7.2 using an 800MHz Varian INOVA spectrometer.

Figure S4. 1 H/ 15 N HSQC NMR spectrum of NOX W52F. The spectrum was collected for apo-NOX with a
Trp 52 to Phe mutation at 50°C in 50mM potassium phosphate buffer at pH 7.2 and using an 800MHz
Varian INOVA spectrometer.

Figure S5. Overlay of NOX spectra obtained for NOX in the apo form (black) and saturated with either
FMN (red) or FAD (blue). Data were collected at 50°C in 50mM potassium phosphate (pH 7.2) with 2.4 mM FMN or 20 mM FAD using an 800MHz Varian INOVA spectrometer.

Figure S6. NMR spectrum of NOX obtained with urea at 20

C. Data were collected with a Varian INOVA spectrometer operating at 800 MHz and 20°C on a NOX sample containing 2.4mM FMN, 50mM potassium phosphate buffer (pH 7.2) and 2.0 M urea.

Figure S7. Backbone ribbon diagrams of NOX 1 (PDB 1NOX), with the two subunits colored in blue and white and the FMN cofactor illustrated in green balls and sticks. In (a), dark purple spheres correspond to residues whose 1 H/ 15 N peaks are missing in spectra of apo NOX but not in the presence of FMN or
FAD. In (b-e) spheres correspond to residues with larger than average 1 H/ 15 N chemical shift perturbations (ppm) in response to changing environmental variables: FMN vs FAD [>0.25 dark purple;
0.17-0.25 purple; 0.11-0.17 dark pink; 0.10-0.11 light pink] (b) 25-50°C [|∂

N
/∂T| > 17 ppb K -1 dark pink;
∂

H
/∂T < -4.5 ppb K -1 light pink; both [|∂

N
/∂T| > 17 ppb K -1 and ∂

H
/∂T < -4.5 ppb K -1 dark purple] (c) and
0 vs 2 M urea [>0.55 dark purple; 0.45-0.45 purple; 0.35-0.45 dark pink; 0.32-0.35 light pink] (d). In (e), residues with Lipari-Szabo order parameters, S 2 , that change upon addition of urea are indicated with spheres, with those that become more rigid shown in light pink while those that become more flexible are shown in dark purple. Malleable regions listed in Table II are labeled on the structure. Image generated with UCSF Chimera.
2

Figure S8. X-ray crystal structure of NOX with FMN (PDB 1NOX 1 ) with elements of secondary structure
and important structural features labelled.

Figure S9. X-ray crystal structures of NOX showing bound to FMN (left) and FAD (right).
1 Spheres correspond to residues with larger than average 1 H/ 15 N chemical shift perturbations (ppm) between
HSQC spectra of NOX with FMN and NOX with FAD [>0.25 dark purple; 0.17-0.25 purple; 0.11-0.17 dark pink; 0.10-0.11 light pink].

Figure S10. Schematic diagram of the proposed gating mechanism. W47 (blue) alternates between an
“open” conformation in which its indole side chain contacts helix F and a water molecule bridges its backbone amide and the FMN cofactor (green), and a “closed” conformation in which its side chain packs more closely against the cofactor and the bridging water molecule is displaced. Shown is the open conformation (1NOX 1 ). Image generated by Chimera.
2

References
1. Hecht HJ, Erdmann H, Park HJ, Sprinzl M, Schmid RD (1995) Crystal structure of NADH oxidase from Thermus thermophilus . Nat Struct Biol 2:1109-1114.
2. Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E, Ferrin T (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25:1605-1612.