MULTIPLE ROTAMERS OF ,2,5,5-TETRAMETHYL-1-OXOPYRROLINYL)-2-PROPEN-1-OL, A STEREOSPECIFIC SUBSTRATE OF LIVER ALCOHOL-DEHYDROGENASE - DETERMINATION OF MOLECULAR-STRUCTURE AND CONFORMATION BY ELECTRON NUCLEAR DOUBLE-RESONANCE

The molecular structure and conformation of the nitroxyl spin-label 2, 2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propen-1-ol has been determined b y electron nuclear double resonance (ENDOR) spectroscopy and molecular modeling. From ENDOR spectra of selectively deuterated analogues of t he spin-labeled propenol in frozen solution, we have assigned resonanc e absorption features for each proton of the propenol side chain. From analysis of the dependence of the ENDOR spectra on H-0, we have ident ified the principal hyperfine coupling (hfc) components for each proto n; from these couplings the dipolar hfc components were calculated in order to estimate electron-proton distances. Torsion angle search calc ulations were carried out to determine the conformational space compat ible with both hard-sphere nonbonded constraints and ENDOR-determined electron-proton distance constraints. Molecular graphics analysis reve aled that the double bond of the side chain is coplanar with the spin- label oxypyrrolinyl ring, exhibiting a single preferred structure, whi le the -CH2OH group exhibits multiple conformers due to rotation aroun d the terminal C-C bond and the C-O bond. We also demonstrate that 5,5 -tetramethyl-1-oxypyrrolinyl)-2-(1-H-2)propenal is catalytically reduc ed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1.) to the spin-labeled propenol in a stereospecific manner.