Proton NMR investigation of heme and surrounding proton in low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla

¹H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla (VtHb-CN) are reported. The assignments of the ¹H NMR spectra of VtHb-CN have been made through MCOSY, NOESY, 1D TOE and SUPERWEFT experiments. Almost all resonance peaks of heme and ligated His85 are identified. The spin-lattice relaxation time T₁'s and the variation relationships of chemical shifts of these peaks with temperature have been acquired, from which the distances between the measured protons and Fe³⁺, and the diamagnetic chemical shifts have been acquired, respectively. The ionization constants of pK_a's of ligated His85 are determined through pH titration of chemical shift, which is 4.95 for ligated His85 C₂H proton. The lower pK_a is attributed to the influence of the Fe³⁺ of carrying positive charge and the coordination of His85 and Fe³⁺ of heme.