Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the N-15 HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Qsites of wild-type and mutant D75H cytochrome ho 3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Delta a, and parameter, delta = vertical bar 2a + T vertical bar - 4v(15N). alpha-strain provides a direct measure of the structural dynamics and heterogeneity of the O center dot center dot center dot H center dot center dot center dot N bond in the SQ systems.