Biophys J, August 2000, p. 1039-1052, Vol. 79, No. 2

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

Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-2436 USA

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.

Biophys J, August 2000, p. 1039-1052, Vol. 79, No. 2
© 2000 by the Biophysical Society   0006-3495/00/08/1039/14  $2.00

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