Figure 1.
Initial velocity (see Methods) is plotted vs. substrate concentration. A. NADH at constant [BQ] = 50 µM. B. BQ at constant [NADH] = 50 µM. C. DCPIP at constant [NADH] = 50 µM. Each plot depicts three temperature treatments of WrbA prior to assay (see text): squares, 5°C; triangles, 23°C; circles, 5°C after 23°C. Solid lines are intended only to guide the eye and do not represent fits to the data.
Figure 2.
Initial velocities at 5°C are plotted vs. substrate concentration. A. Titration of BQ at [NADH] = 100 µM with no salt (circles), 0.25 M NaCl (squares), and 0.5 M NaCl (triangles). B. Titration of DCPIP at [NADH] = 100 µM; symbols as in panel A. Solid lines are intended only to guide the eye and do not represent fits to the data.
Table 1.
Kinetic constants of WrbAa.
Figure 3.
Initial velocities were determined at 23°C to limit the reaction to a single kinetic phase as much as possible. A. Titration of NADH at [BQ] = 10 µM (open squares), 20 µM (triangles), 50 µM (circles), 100 µM (filled squares). B. Titration of BQ at [NADH] = 10 µM (open squares), 20 µM (triangles), 50 µM(circles), 100 µM(filled squares). Solid lines represent non-linear least-squares best fit of the Michaelis-Menten equation to the data points. The values of apparent Km and apparent Vmax returned from the fit are given in Table 1.
Table 2.
Product Inhibitiona.
Figure 4.
A. NADH binding to 50 µM apoWrbA determined by UV spectroscopy. Difference absorbance at 265 nm (see text) is plotted vs. [NADH]. The solid line is intended only to guide the eye and does not represent a fit to the data. B. NAD binding to 200 µM apoWrbA detected by 31P NMR. Spectra at 100, 200, 500, 1000 and 2000 µM NAD from bottom to top, respectively, are overlaid. The bracket with four arrows indicates the doublet pair characteristic of free NAD.
Figure 5.
Each panel shows the sedimentation velocity profile using the whole boundary g(s*) approach of Stafford [28] for apoWrbA (black), WrbA+50 µM FMN (red), and WrbA+50 µM FMN+0.5 mM NAD (blue). A, 3 µM total protein (monomer) at 5°C; B, 3 µM total protein (monomer) at 20°C; C, 20 µM total protein (monomer) at 5°C; D, 20 µM total protein (monomer) at 20°C.
Figure 6.
Substrate binding sites. A. NADH.
View of the active site with NADH bound in the optimized position found by docking as described in the text. Green, molecular surface of holoWrbA calculated from the 2.05 Å crystal structure (PDB ID 3B6J) after removal of the FMN cofactor. Oxidized FMN is depicted as a skeletal model in atomic colors with cyan carbon, and docked NADH with white carbons for differentiation from FMN. Dashed lines represent the indicated distances in Å between nicotinamide C4 and each indicated electron acceptor site of FMN. B. Mutual exclusivity of NADH and BQ. Viewpoint of the binding cavity as in panel A but slightly zoomed out to better depict the steric environment of the full pocket. Translucent white indicates the molecular surface of NADH in the position identified by docking as in panel A; red indicates the molecular surface of BQ calculated from the 1.99 Å crystal structure of the BQ/WrbA complex (PDB ID 3B6K). The part of each substrate that is occluded by the other is represented by the overlap between the red and translucent white surfaces.
Figure 7.
View of the active site residues in direct contact with bound NADH in the optimized docked position. Oxidized FMN is depicted as a skeletal model in red; NADH is depicted as a skeletal model in atomic colors with translucent electrostatic potential surface shaded from red (negative charge) to blue (positive charge). Residues contacting NADH from monomer A are in light blue, from monomer C in green and from monomer D in yellow. A, tetramer. B, dimer.