The binding of substrate/product or transition-state intermediates modifies the properties of medium-chain fatty acyl-CoA dehydrogenase (MCAD) by causing the redox potential to shift positive and the oxygen reactivity to slow by 3000-fold. Two ligands, identified as being the most effective in slowing oxygen reactivity, were 2-azaoctanoyl-CoA and 3-thiaoctanoyl-CoA [Wang, R., & Thorpe, C. (1991) Biochemistry 30, 7895-7901]. We have measured the potential shifts caused by the binding of both ligands to determine which is most similar to the potential shift caused by substrate/product mixture, the assumption being that the best transition-state structural intermediate would give the potential shift most similar to that of substrate/product [Lenn, N.D., Stankovich, M.T., & Liu, H. (1990) Biochemistry 29, 10594-10602]. Both ligands shifted the potential positive, but the shift caused by 2-azaoctanoyl-CoA was 65% that of substrate/product, while 3-thiaoctanoyl-CoA was only 20% of that value. This positive shift is proposed to be caused by a resonance form stabilized by the interaction of the catalytically essential carbonyl of the acyl-CoA with two hydrogen bonds from the enzyme, which induces a partial negative charge on the carbonyl and a partial positive charge on carbon 2 of the ligand and carbon 3 of the substrate/product couple. The X-ray structure shows that carbons 2 and 3 of the substrate/product overlap the diazadiene portion of the flavin ring [Kim, J.-J. P., Wang, M., & Paschke, R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7523-7527].(ABSTRACT TRUNCATED AT 250 WORDS)