Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.