Figure 1.
Substrate inhibition by NADH in an ordered bireactant mechanism.
A. Double-reciprocal plots of initial velocities versus substrate concentrations assayed with fixed concentration of NADH: 9.5 µM (open diamond), 19 µM (closed diamond), 25 µM (open triangle), 37.5 µM (closed triangle), 50 µM (open square), 75 µM (closed square), 100 µM (open circle), and 200 µM (closed circle). The VMax is calculated based on the y-axis intercept on this plot. B. Relationship between the slopes (i.e., Slope 1/CrO42−) in Figure 1A at each of seven fixed NADH concentrations. C. Double-reciprocal plots of initial velocities versus substrate concentrations with fixed concentration of CrO42−: 31 µM (open triangle), 62 µM (closed triangle), 125 µM (open square), 250 µM (closed square), 500 µM (open circle), and 1000 µM (closed circle). At low NADH concentrations it is possible to fit the data with a straight line. However, at high NADH concentrations, individual curves bend upwards. Values for KmA, KmB, Kia and Ki were calculated from axes-intercepts and slopes in panels B and C (see Table S2) [20]. D. Cleland notation depicting catalytic mechanism of Gh-ChrR, showing substrate inhibition by NADH binding to FMN-E to form a dead-end complex FMN-E-NADH that competes with metal complex formation, Mox-FMNH2-E-NADH.
Table 1.
Data Collection and Structural Refinement Statistics of Gh-ChrR.
Figure 2.
Monomeric (A) and tetrameric (B) depictions of the 2.25 Å structure of Gh-ChrR showing the backbone fold, a space-filling model of bound FMN (elements color: red = oxygen, blue = nitrogen, gray = carbon) and bound chloride anion (green sphere). Secondary structural elements including the 310 helices (η) are numbered sequentially from the N-terminus. C. Electrostatic potentials at the solvent-accessible surface of Gh-ChrR. A stick model of the FMN molecule and the associated chloride ion (gray sphere) is highlighted. The electrostatic potential are drawn (Pymol) at a level of −71.817 kT/e (red) to +71.817 kT/e (blue), where k is the Boltzman’s constant, T is the absolute temperature, and e is the magnitude of the electron charge.
Figure 3.
Structure proximal to bound FMN.
A. Electron density surrounding FMN and chloride ion (gray sphere) contoured at 1.0 σ. B. Schematic representation of hydrophobic contacts (arc with radiating spokes) and potential hydrogen bonds (dashed lines) between FMN and two monomeric units (chain A and C) of the Gh-ChrR tetramer. Atoms are color-coded: black = carbon, red = oxygen, blue = nitrogen. This image was produced using the program LIGPLOT [62].
Figure 4.
Putative Gh-ChrR NADH and substrate binding sites.
A. NADH was modeled into the Gh-ChrR structure by superimposing it with the NADH-containing structure of EmoB (PDB entry: 2VZJ, Figure S7). The nicotinamide ring of NADH (primarily green stick model) is stacked on top of the isoalloxazine ring of FMN (primarily yellow stick model), and the adenosine part of NADH points to ribtyl group of FMN. The black arrow indicates the distance from C4N of NADH to the si-face of the FMN isoalloxazine ring. Residues N53, D54, E57, S100, R101 and F137 from chain A (cyan) and residues N85, P119, and T154 from chain C (gold) interact with NADH. B. The putative active site of Gh-ChrR shown with bound FMN (primarily yellow stick model) and a chloride ion (green sphere). The black arrow indicates the distance from the Cl− to the si-face of the FMN isoalloxazine ring. Key residue R101 holding chloride ion in place is shown in a stick model. Critical residues for hydride transfer, N85 and Y86 from chain A (cyan) and S118 from chain C (gold) are shown in a stick model. The green dash lines indicate the distance (∼3 Å) between N of amide group of N85/Y86 and O4, and the distance (∼3 Å) between OG of hydroxyl group of S118 and O2.
Table 2.
Catalytic Influence of Site-Directed Substitution of Putative Metal and Cofactor Ligands on NADH-Dependent Chromate Reduction Efficiency.