INSIGHTS FROM TIMS-MS, IRMPD SPECTROSCOPY, AND MOLECULAR DYNAMICS ON NICOTINAMIDE ADENINE DINUCLEOTIDE STRUCTURAL DYNAMICS: NAD⁺ VS NADH

Juan Camilo Molano-Arevalo¹; Walter Gonzales¹; Kevin Jeanne Dit Fouque¹; Jaroslava Miksovska^1,2; Phillippe Maitre³, and Francisco Fernandez-Lima^1,2*.

1 Department of Chemistry and Biochemistry, Florida International University, Miami, USA
2 Biomolecular Sciences Institute, Florida International University, Miami, USA
3 Laboratoire de Chimie Physique, Université Paris Sud, UMR 8000 CNRS, Orsay Cedex 91405, France

Nicotinamide adenine dinucleotide is found in all living cells where the oxidized (NAD⁺) and reduced (NADH) forms play important roles in many enzymatic reactions; however, little is known about NAD⁺ and NADH conformational changes and kinetics as a function of the cell environment. In the present work, NAD⁺ and NADH were studied as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution, time decay studies showed a fast and slow component and dependence on the organic content. NAD gas-phase studies permitted the characterization of NAD⁺ and NADH structures and conformers using TIMS-MS and IRMPD. Two ion mobility bands were observed for the protonated and sodiated NAD⁺ species, while four and two ion mobility bands for the protonated and sodiated NADH species, respectively. IRMPD spectroscopy of NAD⁺ and NADH protonated species showed common and signature bands between the NAD forms. Results show that NAD⁺ and NADH species exist in an open, stack, and closed conformations and that the driving force for conformational dynamics is the interaction between the adenine and nicotinamide rings.