The nature of the NADP complex with manganese(II) ions as studied by proton and phosphorus magnetic resonance

Abstract

Proton and phosphorus magnetic resonance techniques have been applied to study the conformation of the complex formed between Mn(II) ions and nicotinamide adenine dinucleotide phosphate (NADP) in aqueous solutions at a pH of 7.5 and 20 °C. The dipolar correlation time (τc) for the Mn(II)–NADP complex at 20 °C was calculated to be 1.7 × 10−10 s, based on the measurement of proton longitudinal relaxation time ratios and a literature value of τc for nicotinamide adenine dinucleotide (NAD). The τc, as well as the 1H and 31P relaxation data together with the measured stability constant for this complex, were used to calculate the distances from the 1H and 31P nuclei to the Mn(II) ion. These distances are 3.8 Å to A8, 5.0 Å to A2, 4.5 Å to A1′, 5.3 Å to N2, 5.4 Å to N6, 5.5 Å to N5, 5.8 Å to N1′, and 6.3 Å to N4. The distances to the linkage 31P nuclei are 3.7 Å, and 3.3 Å to the adenosine ribose 2′-phosphate. Based on these distances, a model is proposed for the Mn(II)–NADP complex. In this complex, the Mn(II) ion is bonded to the oxygens of all three phosphates, and the adenine and nicotinamide bases are 7–8 Å apart and parallel. Hence the Mn(II) ion is located between and to one side of the two bases but is distinctly closer to the adenine base. The adenine base is in the anti conformation, whereas the nicotinamide moiety can be either syn or anti. Hence there is little or no close stacking of the two bases in the complex. The addition of Mg(II) ions to a solution of free NADP caused no observable shift of the aromatic proton peaks, indicating no differences in shielding effects. Hence the rings are probably similarly oriented in both free NADP and the metal–NADP complex. Effects on the transverse relaxation rates due to the paramagnetic metal ion (T2p−1) were also measured from the 31P linewidths as a function of temperature. The lifetime of the metal ion in the complex is 4.0 ± 0.5 × 10−6 s and ΔH≠ for the exchange process is 13.7 ± 0.5 kcal/mol.