Mechanistic Analysis of Fluorescence Quenching of Reduced Nicotinamide Adenine Dinucleotide by Oxamate in Lactate Dehydrogenase Ternary Complexes

Abstract

Fluorescence of Reduced Nicotinamide Adenine Dinucleotide (NADH) is extensively employed in studies of oxidoreductases. A substantial amount of static and kinetic work has focused on the binding of pyruvate or substrate mimic oxamate to the binary complex of lactate dehydrogenase (LDH)-NADH where substantial fluorescence quenching is typically observed. However, the quenching mechanism is not well understood limiting structural interpretation. Based on time-dependent density functional theory (TDDFT) computations with cam-B3LYP functional in conjunction with the analysis of previous experimental results, we propose that bound oxamate acts as an electron acceptor in the quenching of fluorescence of NADH in the ternary complex, where a charge transfer (CT) state characterized by excitation from the highest occupied molecular orbital (HOMO) of the nicotinamide moiety of NADH to the lowest unoccupied molecular orbital (LUMO) of oxamate exists close to the locally excited (LE) state involving only the nicotinamide moiety. Efficient quenching in the encounter complex like in pig heart LDH requires that oxamate forms a salt bridge with Arg-171 and hydrogen bonds with His-195, Thr-246 and Asn-140. Further structural rearrangement and loop closure, which also brings about another hydrogen bond between oxamate and Arg-109, will increase the rate of fluorescence quenching as well.

Figure 1

Jablonski diagram of the photodynamics of NADH. Some reported lifetimes are shown for selected excited states. Ad = adenylyl group; NA = dihydronicotinamide group.

Figure 2

X-ray crystal structural information of the active sites for phLDH (top, PDB ID: 9LDB) and bsLDH (bottom, PDB ID: 1LDN). Distance unit: Å. Here the structure of pig muscle LDH is used since pig heart LDH structure (PDB ID: 5LDH) does not have oxamate. It should be noted that the structure of LDH-NADH-oxamate of pig muscle is very similary with that of pig heart (unpublished results).

Figure 3

Different views of pruned structures from x-ray crystallographic structure (PDB ID: 9LDB). Residues form hydrogen bonds with oxamate include Arg-171, Thr-246, Asn-140, His-195, and Arg-109. Residues involve hydrogen bonds with dihydronicotinamide are Val-138, Ser-163, Gly-164, and a water molecule. Asp-168 is also included because it interacts with His-195 and neutralizes the positive charge on it. NA = 1-methyl-1,4-dihydronicotinamide.

Figure 4

Stern-Volmer plots of fluorescence quenching of NADH by acrylamide, pyruvate, and oxamate.

Figure 5

Electrostatic potential surface of phLDH(PDB ID: 9LDB). It is clearly seen that the cavity around oxamate and the nicotinamide moiety of NADH is positively charged.

Figure 6

Structures of selected known NADH fluorescence quenchers (left) and enhancers (right).

Figure 7

Structural description of steric effect induced by substrates.

Figure 8

Fluorescence of NADH in solution (dashed line) and binary complex (phLDH/NADH) (solid line) at pH 6 buffer. Excited at 340 nm. [NADH] = 10 μM, [phLDH] = 22 μM.

Figure 9

Sample frontier orbitals involved in the locally excited (LE) states and charge transfer (CT) states. The LE state will refer to π-π* transition of nicotinamide. The CT state will refer to π, nicotinamide-π*, oxamate transition.

Figure 10

Energetic comparison of LE and CT states in selected cases in vacuum.