STRUCTURAL MODULATION OF 2-ENOYL-COA BOUND TO REDUCED ACYL-COA DEHYDROGENASES - A RESONANCE RAMAN-STUDY OF A CATALYTIC INTERMEDIATE

A catalytic intermediate, the so-called ''purple complex,'' of acyl-Co A dehydrogenase is produced on its reaction with the substrate, acyl-C oA. The purple complex is a charge-transfer complex between the reduce d enzyme and the product, enoyl-CoA. Resonance Raman spectra of the pu rple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydro genases] were measured with excitation at 632.8 nm within charge-trans fer absorption bands. The 1,577 cm(-1) band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1)=0 stretching of crotonyl-CoA, judging from the isotopic frequenc y shifts upon C-13 Or O-18 substitution of butyryl-CoA. The 1,627 cm(- 1) band of the C(1)=0 moiety of crotonyl-CoA in solution shifted downw ard by 50 cm(-1) on complexation with reduced SCAD. This large frequen cy shift indicates a substantial interaction between C(1)=0 and the en zyme, and is further evidence for an appreciable contribution of a pol arized form of the C(1)=0 moiety in the enzyme-bound enoyl-CoA. This f requency shift can be explained by the hydrogen bond of C(1)=0, The 1, 577 cm(-1) band of the MCAD purple complex remained constant, regardle ss of the acyl carbon-chain length (from C4 to C16 of the substrate, a cyl-CoA); the alkyl chain scarcely affected the interaction of the C(1 )=0 moiety in the active site. The frequency of the 3-methylcrotonyl-C oA carbonyl C(1)=0 moiety shifted from 1,628 cm(-1) in solution downwa rd by 45 cm(-1) when the CoA thioester bound to reduced IVD, but by 28 cm-l when the thioester bound to reduced SCAD or MCAD. This indicates that the hydrogen bond at C(1)=0 of 3-methylcrotonyl-CoA in SCAD or M CAD is weaker than in the case of IVD; the steric repulsion of the 3-m ethyl group probably changes the orientation of the -C(3)H=C(2)H-C(1)= 0 moiety and thus affects the hydrogen bonding, Tyr-375, which is cons erved in straight chain acyl-CoA dehydrogenases, may be responsible fo r the steric repulsion and thus play a role in the substrate specifici ty.