Interspin distances in spin-labeled metmyoglobin variants determined by saturation recovery EPR

Saturation recovery (SR) electron paramagnetic resonance was used to determ ine the distance between iron and nitroxyl for spin-labeled metmyoglobin va riants in low-spin and high-spin states of the Fe(III). The interspin dista nces were measured by analyzing the effect of the heme iron on the spin-lat tice relaxation rates of the nitroxyl spin label using the modified Bloembe rgen equation for low-spin species, and an analogue of the Bloembergen equa tion for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distan ces obtained by SR for high-spin and low-spin complexes with 15-20 Angstrom interspin distances, for low-spin CN- and high-spin formate adducts at dis tances up to about 30 Angstrom, and results from Insight calculations were in good agreement. For variants with 25-30 Angstrom interspin distances, th e distances obtained by SR for the fluoride adducts were shorter than obser ved for the CN- or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN-, imidazole, F-, and formate), CN- is t he best choice for determination of iron-nitroxyl distances in the range of 15-30 Angstrom.