Interspin Distances in Spin-Labeled Metmyoglobin Variants Determined by Saturation Recovery EPR

Abstract

Saturation recovery (SR) electron paramagnetic resonance was used to determine the distance between iron and nitroxyl for spin-labeled metmyoglobin variants in low-spin and high-spin states of the Fe(III). The interspin distances were measured by analyzing the effect of the heme iron on the spin-lattice relaxation rates of the nitroxyl spin label using the modified Bloembergen equation for low-spin species, and an analogue of the Bloembergen equation for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distances obtained by SR for high-spin and low-spin complexes with 15–20 Å interspin distances, for low-spin CN − and high-spin formate adducts at distances up to about 30 Å, and results from Insight calculations were in good agreement. For variants with 25–30 Å interspin distances, the distances obtained by SR for the fluoride adducts were shorter than observed for the CN − or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN −, imidazole, F −, and formate), CN − is the best choice for determination of iron-nitroxyl distances in the range of 15–30 Å.