| The determination of interspin distances in proteins and polymers by electron paramagnetic resonance provides crucial long-distance structural constraints. The goal of this work is to compare electron-electron interspin distances determined from enhancement by rapidly relaxing hemes of spin-lattice and spin-spin relaxation of nitroxyls.; Thirteen sperm whale myoglobin (Mb) variants (Hl2C, A15C, A19C, A53C, K56C, A57C, V66C, K87C, Q91C, K98C, E148C, L149C, and G150C) were expressed in a bacterial system as apomyoglobin. The proteins were isolated from inclusion bodies under denaturing conditions, refolded, spin labeled with (1-oxyl-2,2,5,5-tetramethyl-Delta 3-pyrroline-3-methyl)methanethiosulfonate (MTSL), and reconstituted with hemin. Exogenous axial ligands were CN- for low spin heme and F- for high-spin heme.; Spin lattice relaxation rates, 1/T1, for nitroxyl and heme iron were measured in the absence of spin-spin interaction. Iron-nitroxyl interspin distances were determined by analyzing saturation recovery curves for the nitroxyl at multiple temperatures, using locally-written software that is based on the Bloembergen equation. The distances for the variants were 18 to 30 A. The distances determined for high spin and low spin forms for the same variant agreed with each other within experimental error, and with the results of molecular modeling using the Insight IIRTM program.; Nitroxyl 2-pulse electron spin echo decay curves, and the temperature dependence of relative echo intensities were analyzed using locally written software based on the analysis of Zhidomirov and Salikhov. The agreement between the experimental and calculated echo decay curves and of relative echo intensity as a function of temperature is better below about 25 K for low spin variants, and 40 K for high spin variants than at higher temperatures, which indicates that an additional contribution to echo dephasing is present. The distances determined by electron spin echo spectroscopy for low spin variants agreed well with the distances determined by saturation recovery and Insight simulations in the range of distances studied. The very large impact of high-spin Fe(III) on echo dephasing suggests that it will be more useful for interspin distance determination at distances longer than about 25 A. |
