¹H nuclear relaxation study of the aniline-binding site in human hemoglobin

Hemoglobin-catalyzed hydroxylation of aniline may be taken as a model for similar reactions catalyzed by cytochrome P-450. Using ultraviolet-difference spectroscopy and ¹H nuclear relaxation techniques, the binding of aniline to hemoglobin was examined. From the magnitude of paramagnetic effects of ferric iron on aniline protons, using the correlation time determined from the magnetic field dependence of water proton relaxation rates, aniline was found to bind to methemoglobins such that the aromatic protons are 8.5 ± 0.7 Å, away from the high-spin Fe³⁺. A mode of binding is proposed where the aniline molecule is hydrogen bonded to the distal histidine of hemoglobin.