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      Role of Aromatic Stacking Interactions in the Modulation of the Two-Electron Reduction Potentials of Flavin and Substrate/Product inMegasphaera elsdeniiShort-Chain Acyl-Coenzyme A Dehydrogenase†

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      Biochemistry
      American Chemical Society (ACS)

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          Abstract

          The effects of aromatic stacking interactions on the stabilization of reduced flavin adenine dinucleotide (FAD) and substrate/product have been investigated in short-chain acyl-coenzyme A dehydrogenase (SCAD) from Megasphaera elsdenii. Mutations were made at the aromatic residues Phe160 and Tyr366, which flank either face of the noncovalently bound flavin cofactor. The electrochemical properties of the mutants were then measured in the presence and absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox studies suggest that the phenylalanine and tyrosine both engage in favorable pi-sigma interactions with the isoalloxazine ring of the flavin to help stabilize formation of the anionic flavin hydroquinone. Disruption of these interactions by replacing either residue with a leucine (F160L and Y366L) causes the midpoint potential for the oxidized/hydroquinone couple (E(ox/hq)) to shift negative by 44-54 mV. The E(ox/hq) value was also found to decrease when aromatic residues containing electron-donating heteroatoms were introduced at the 160 position. Potential shifts of -32 and -43 mV for the F160Y and F160W mutants, respectively, are attributed to increased pi-pi repulsive interactions between the ring systems. This study also provides evidence for thermodynamic regulation of the substrate/product couple in the active site of SCAD. Binding to the wild-type enzyme caused the midpoint potential for the butyryl-CoA/crotonyl-CoA couple (E(BCoA/CCoA)) to shift 14 mV negative, stabilizing the oxidized product. Formation of product was found to be even more favorable in complexes with the F160Y and F160W mutants, suggesting that the electrostatic environment around the flavin plays a role in substrate/product activation.

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          Author and article information

          Journal
          Biochemistry
          Biochemistry
          American Chemical Society (ACS)
          0006-2960
          1520-4995
          June 2001
          June 2001
          : 40
          : 25
          : 7720-7728
          Article
          10.1021/bi010206s
          11412126
          ddac68fb-1f07-424c-88a3-83a12a769a51
          © 2001
          History

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