An electron spin resonance spectral study of the dynamics of spin-labelled fatty acids bound to oxidoreductases
Electron spin resonance (ESR) spectra have been recorded for 5-doxylstearate (I) and 16-doxylstearate (II) in the presence of bacterial luciferase and soybean lipoxygenase. The acids are inhibitors of the enzymes (Kd ≈ 2 × 10−5 M for II bound to luciferase). Using theoretical computer simulations of the ESR line shapes, an effective correlation time of τeff ≈ 3.7 × 10−9 ± 0.5 × 10−9 s is found for the motion of spin label I bound to luciferase. Concentration-dependent sedimentation velocity experiments indicate luciferase is anisotropic and, assuming a prolate ellipsoid of axial ratio ≤ 9, a correlation time of τa ≈ 7.7 × 10−9 s is predicted for rotation about the luciferase long axis. The tightly bound spin-labelled inhibitors, therefore, are proposed to give rise to ESR spectra chiefly reflecting luciferase long axis rotation. The ESR spectra of I and II, bound to lipoxygenase and to luciferase, appear similar.