Cyclic Changes in Active Site Polarization and Dynamics Drive the 'Ping-pong' Kinetics in NRH:Quinone Oxidoreductase 2: An Insight from QM/MM Simulations

Abstract

Quinone reductases belong to the family of flavin-dependent oxidoreductases. With the redox active cofactor, flavin adenine dinucleotide, quinone reductases are known to utilize a 'ping-pong' kinetic mechanism during catalysis in which a hydride is bounced back and forth between flavin and its two substrates. However, the continuation of this catalytic cycle requires product displacement steps, where the product of one redox half-cycle is displaced by the substrate of the next half-cycle. Using improved hybrid quantum mechanical/molecular mechanical simulations, both the catalytic hydride transfer and the product displacement reactions were studied in NRH:quinone oxidoreductase 2. Initially, the self-consistent charge-density functional tight binding theory was used to describe flavin ring and the substrate atoms, while embedded in the molecular mechanically-treated solvated active site. Then, for each step of the catalytic cycle, a further improvement of energetics was made using density functional theory-based corrections. The present study showcases an integrated interplay of solvation, protonation, and protein matrix-induced polarization as the driving force behind the thermodynamic wheel of the 'ping-pong' kinetics. Reported here is the first-principles model of the 'ping-pong' kinetics that portrays how cyclic changes in the active site polarization and dynamics govern the oscillatory hydride transfer and product displacement in this enzyme.

Keywords: Double displacement reactions; Kohn-Sham density functional theory; flavoenzyme; hydride transfer reactions; quantum mechanical/molecular mechanical calculations; quinone oxidoreductase 2; self-consistent charge density functional tight-binding theory; ‘ping-pong’ kinetics.

Figure 1.

The reaction coordinates of the two hydride transfer reactions involving 7, 8 dimethylisoalloxazine (flavin) as defined in eq. 6: a) from NMH to flavin ring and b) flavin to PBQ. The color of the reduced and oxidized systems are coded with blue and red, respectively. The ‘R’ attached to the flavin ring is a methyl group for aqueous systems and represents a −ribityl-ADP moiety for the enzymatic reactions.

Figure 2.

Corrected potentials of mean force (PMF) for the two hydride transfer processes catalyzed in both enzyme-bound (solid line) and aqueous (dashed line) states: a) NMH to oxidized flavin and b) reduced flavin to PBQ. For each catalytic reaction, the reaction coordinate ξ is calculated using eq. 6 and illustrated in Figure 1.

Figure 3.

Hydrogen bonding interactions for the transition state structures observed in the two hydride transfer reactions. The substrates are stacked on top of the tricyclic isoalloxazine ring: a) the hydride donor substrate, NMH and b) the hydride acceptor substrate, PBQ. Selected interactions with the surrounding side chains are highlighted using purple broken lines.

Figure 4.

Iso-electron density surfaces of the PC and SC for the hydride transfer reactions: NMH to oxidized flavin and the reduced flavin to PBQ. Each surface is color coded based on their natural bond orbital-computed partial charges: red for negative charges and green for positive charges. In each structure, the dipole moment, μ is shown from the center of each complex by a blue arrow.

Figure 5.

The radial distribution function (RDF) of water around the QM site for the PC (blue line) and the SC (green line) in the catalytic hydride transfer reactions involving a) NMH and b) PBQ. Dotted illustrations in c) indicates the accumulation of water molecules during around the QM region. The O2, N5, and C8 atoms of the NQO2-bound flavin were chosen to compute the RDFs. Data from 500 ps dynamics simulation was used for all calculations and visualizations.

Figure 6.

Computed energetic and geometric changes of selected charged residues in the catalysis and product displacement reactions of ‘ping-pong’ cycle as shown in Scheme 1 (reactions a–d). The ΔE_elec for each charged residue, computed using eq. 9, averaged over 500 ps data, provides a measure of the electrostatic stabilization energy of the QM region defined in Scheme 2. The ΔE_elec values were plotted against the change in the C_α− flavin COM distances. Charged residues in a) are labeled by the same color as the arrows that represent the change in geometrtric and energetics for the specific process in Scheme 1. A positive value (i.e. upward arrow) of ΔE_elec indicates that the residue favored the specific reaction step. An arrow pointing to the right signifies that the C_α atom moved further away from the QM region.

Figure 7.

The essential dynamics of the NQO2 active site during the hydride transfer reactions. The left and right panel shows the principal component of motions during the hydride transfer reactions involving NMH and PBQ, respectively. A cartoon representation of the holo-enzyme is shown in the middle, the four dynamic regions identified in our earlier studies are highlighted: helix (191–217) of subunit A as blue, loop (149–165) of subunit A as red, loop (125–137) of subunit B as green, and loop (55–78) of subunit B as purple. The arrows of the dynamic regions in the left and right panels are color coded to match with the structural elements of the holo-enzyme (central panel). The conformational changes in the left and right panels are indicated by superimposed backbones shown in tubes: the initial conformation in red and the final one in blue.

Scheme 1.

The ‘ping-pong’ kinetics in NQO2 comprises two redox half cycles, each of which consists of a catalytic hydride transfer step (steps a or c) and a product displacement step (steps b or d). The first half cycle starts with step a), during which a hydride (shown in green color) is transferred from the NMH (in blue) to the neutral flavin. This is followed by a product displacement step b), when the oxidized product is displaced by PBQ (in red). In the next half cycle, during step c), a hydride is transferred from the reduced flavin to the PBQ. Subsequently, in step d) the protonation (the proton shown in purple) of the anionic hydroquinone and subsequent displacement of the hydroquinone by NMH occurs, thereby completing the cycle.

Scheme 2.

The computational setup for the hybrid QM/MM simulation. The flavin ring of the FAD and the substrates (shown within the elliptical boundary) are inside the QM region, while embedded into a 30 Å spherical region of solvated NQO2 active site. A link-atom shown by ‘L’ acts as the boundary between the QM and MM regions.

Scheme 3.

The protocol used in the study for incorporating quantum corrections to the free energies of reaction for the steps of ‘ping-pong’ kinetics. The free energy obtained in the SCC-DFTB-D/MM was corrected using two corrections derived from the study of small model systems comprising only QM atoms: i) a high-level correction (HLC), obtained from the difference in the DFT and SCC-DFTB-D-computed Born-Oppenheimer potential energies for each model systems; ii) the difference in the DFT-computed zero-point energy corrections (ΔZPE), for each step.

Scheme 4.

The thermodynamic scheme employed for the product displacement reactions. The reaction at the center represent the gas-phase product displacement, while those at the top and bottom show the displacement processes in the aqueous and enzyme environments, respectively. ‘Fl’ is the short-hand notation of the flavin ring, while the ligand S1 is displaced by the ligand S2, in each case. The oxidation states of the flavin as well as the ligands depend on the specific half-cycle of the ‘ping-pong’ kinetics being considered. Such complexes are indicated in the right bottom corner; reduced flavin binds to the oxidized form of nicotinamide and PBQ, while neutral flavin binds to the hydroquinone and NMH.

Scheme 5.

The free energies obtained in this study for the catalytic and product displacement steps of the ‘ping-pong’ kinetics. The red and blue shades indicate the oxidized and reduced states of the flavin-bound active site, respectively. The purple glow indicates a more polarized active site as observed for the product state of both hydride transfer reactions.