Spin label studies on the flavoproteins lipoamide dehydrogenase and D-amino acid oxidase.

Maleimide spin label was covalently bound to sulfhydryl residues of D-amino acid oxidase and lipoamide dehydrogenase. Labeling of native D-amino acid oxidase resulted in a non-homogeneous EPR-spectrum, which consisted of 4 moles of spin label bound immobile to the enzyme (mol.-weight 90.000). A detailed analysis of the spectrum, the kinetics of the reaction of the spin label with the protein and the temperature dependence of the spectrum showed that the spectrum originates from two different pairs of sulfhydryl groups. The spin labeled lipoamide dehydrogenase yielded also a mixed EPR-spectrum of two different bound spin labels, i.e. an immobile and a semimobile species. The temperature dependence gave for both types of spectra a transition point at 10 °C. Titration with urea gave only for the immobile species a transition at 1.5 M. Part of the semimobile species seems to be bound near the active site. D-amino acid oxidase was also specifically labeled, near the active site, with a substrate analogue. From its EPR-spectrum it appeared that the analogue was bound very mobile (r c = 0.3nsec) with respect to the protein. Removal of FAD had a drastic reversible effect on the spectrum.