(1)H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla (VtHb-CN) are reported. The assignments of the(1)H 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 T (1)'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 Fe(3+), 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 C(2)H proton. The lower pK (a) is attributed to the influence of the Fe(3+) of carrying positive charge and the coordination of His85 and Fe(3+) of heme.