Interspin distances determined by time domain EPR of spin-labeled high-spin methemoglobin

Abstract

Continuous wave (CW) EPR spectra of the nitroxyl signal in high-spin fluoro- and aquo-methemoglobin spin-labeled at the β-93 cysteines exhibited line broadening as the temperature was reduced from 20 to 5 K. In the temperature interval where the CW spectra exhibited broadening, electron spin echo data showed a dramatic increase in the phase memory relaxation rate, 1/ T m, for the nitroxyl. The lineshape changes and changes in 1/ T m are attributed to iron—nitroxyl interaction in the regime where the iron relaxation rate in s −1 is of the same order of magnitude as the iron—nitroxyl spin—spin splitting expressed in s −1. Saturation recovery data for the nitroxyl signal in spin-labeled fluoro- and aquo-methemoglobin between 15 and 150 K demonstrated that interaction with the high-spin iron(III) enhanced the electron spin relaxation rate of the spin label. Electron spin relaxation rates for the m s = ± 1/2 transitions of the high-spin iron(III) were determined by electron spin echo and inversion recovery at 4.2–6 K and by analysis of the temperature-dependent contribution to the iron CW linewidths between about 20 and 150 K. The saturation recovery data for the nitroxyl in the spin-labeled methemoglobins were analyzed to determine the interspin distances, using a modified version of the Bloembergen equation and experimentally determined iron relaxation rates. Interspin distances in the high-spin methemoglobins (15.8–17.5 Å) are systematically longer than in the low-spin analogs (15–15.5 Å).