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

Yi Zhou, Bruce E. Bowler, Kim Lynch, Sandra S. Eaton, Gareth R. Eaton

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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 Bioembergen 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.

Original languageEnglish
Pages (from-to)1039-1052
Number of pages14
JournalBiophysical Journal
Issue number2
StatePublished - 2000


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