¹⁴N Mims pulsed-ENDOR of proximal histidine and heme of aquometmyoglobin and fluorometmyoglobin

Previous ¹⁹F and ^1.2H electron-nuclear double resonance (ENDOR) study of fluorometmyoglobin (MbF) in frozen-solution state provided sensitive tools sensing subtle structural changes of the heme that are not obtainable from X-ray. [Fann et al., J. Am. Chem. Soc. 1995, 117, 6019] Because of the intrinsic inhomogeneouse EPR line broadening effect of MbF in frozen-solution state, detection of the electronic and geometrical changes of the heme ring itself and the proximal histidine by using ¹⁴N CW ENDOR was interfered. In the present study, hyperfine-sensitive ¹⁴N Mims ENDOR technique of pulsed-EPR was employed to probe the changes. With two different τ values of 128 and 196 ns, ¹⁴N ENDOR signals of the heme and proximal histidine were completely resolved at g′∥ (≈ g_e ≈ 2). This study presents that X-band ¹⁴N Mims ENDOR sequence can sensitively detect the small changes of the spin densities and p orbital populations of the proximal and the heme nitrogens, caused by ligand and pH variation of the distal site.