Deuterium Nuclear Magnetic Resonance Spectroscopy of Deuterated Pyridine-Iron (III) Porphyrin Complexs. Locations and Relaxation Times of Bound Deuterated Pyridine Resonances

Abstract

The first application of deuterium nuclear magnetic resonance spectroscopy (²H NMR) to fully deuterated pyridine (d₅-pyridine)-iron (III) porphyrin complexes is described.
(1) d₅-Pyridine gives very broad ²H NMR signals in the presence of hemin or horseradish peroxidase to which pyridine is hardly (or not) bound, probably due to relatively long electronic relaxation times of the high-spin ferric ions. d₅-Pyridine in the presence of horse-heart metmyoglobin gives resolved and less broadened ²H NMR signals, probably due to the relatively short electronic relaxation times of the ferric ion and/or to slow chemical exchange of the ligand.
(2) The three resonances of free d₅-pyridine coalesce into a single or a double resonance in the presence of a heme octapeptide prepared by trypsin digestion of Candida krusei cytochrome c. Nuclear spin-lattice and spin-spin relaxation times of the d₅-pyridine-heme octapeptide complex are markedly shorter than those of free d₅-pyridine. These findings are interpreted by the chemical exchange mechanism from the temperature dependences of the relaxation times. Thus, on certain assumptions the residence time of pyridine in the bound state and the exchange rate are estimated as _??_10^-3 s and _??_400 s^-1, respectively, at 25°C. Since ¹H NMR signals of axial ligands of paramagnetic hemoproteins are hard to observe, the usefulness of deuterium magnetic resonance for investigating ligand exchange in the paramagnetic hemoproteins is emphasized.