Determination of the distance between the two neutral flavin radicals in augmenter of liver regeneration by pulsed ELDOR.
Pulsed electron-electron double resonance (ELDOR) has been used to obtain structural information from a FAD-dependent sulfhydryl oxidase, Augmenter of Liver Regeneration (ALR). ALR is a homodimer with each subunit containing a noncovalently bound FAD cofactor. Both FADs may be converted into the blue neutral radical form by aerobic treatment with DTT. From three-pulse and four-pulse ELDOR experiments, a distance of 26.1 +/- 0.8 A could be determined between the FAD cofactors in human ALR. Taking into account the electron spin density distribution in a neutral flavin radical obtained from density functional theory calculations, a distance of 26.9 A could be estimated for the separation of the spin centers in the X-ray structure of rat ALR. The good agreement confirms that rat ALR may be used as a model for mechanistic discussions of human ALR. The experiments also demonstrate that neutral flavin radicals have the appropriate properties to be used as intrinsic spin labels for distance determinations in proteins.
Kay
Christopher W M
CW
Institut für Experimentalphysik, Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany. chris.kay@physik.fu-berlin.de
Elsässer
Celine
C
Bittl
Robert
R
Farrell
Scott R
SR
Thorpe
Colin
C
D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005415:Flavins / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D050862:Oxidoreductases Acting on Sulfur Group Donors
D005415:Flavins; D005609:Free Radicals; D010088:Oxidoreductases; D011506:Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C088825:GFER protein, rat