3 years ago

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

Mechanistic Analysis of Fluorescence Quenching of Reduced Nicotinamide Adenine Dinucleotide by Oxamate in Lactate Dehydrogenase Ternary Complexes
Robert Callender, Huo-Lei Peng
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. Oxamate efficiently quenches the fluorescence of NADH in the lactate dehydrogenase (LDH) ternary complex, but not in water, through electron transfer (ET) mechanism. Oxamate interacts with LDH by forming a salt bridge with Arg-171 and hydrogen bonds with His-195, Thr-246 and Asn-140 in the encounter complex. It adds another hydrogen bond with Arg-109 after loop closing. Such interactions decrease the energy of a charge transfer (CT) state involving the nicotinamide moiety and oxamate. Then, electron transfer from excited states of NADH to oxamate and consequent fluorescence quenching take place.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/php.12775

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