Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase
Jd. Pellett et al., Role of aromatic stacking interactions in the modulation of the two-electron reduction potentials of flavin and substrate/product in Megasphaera elsdenii short-chain acyl-coenzyme A dehydrogenase, BIOCHEM, 40(25), 2001, pp. 7720-7728
The effects of aromatic stacking interactions on the stabilization of reduc
ed flavin adenine dinucleotide (FAD) and substrate/product have been invest
igated 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 ele
ctrochemical properties of the mutants were then measured in the presence a
nd absence of a butyryl-CoA/crotonyl-CoA mixture. Results from these redox
studies,suggest that the phenylalanine and tyrosine both engage in favorabl
e pi-sigma interactions with the isoalloxazine ring of the flavin to help s
tabilize formation of the anionic flavin hydroquinone. Disruption of these
interactions by replacing either residue with a leucine (F160L and Y366L) c
auses 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 decrea
se when aromatic residues containing electron-donating heteroatoms were int
roduced at the 160 position. Potential shifts of -32 and -43 mV for the F16
0Y and F160W mutants, respectively, are attributed to increased pi-pi repul
sive interactions between the ring systems. This study also provides eviden
ce for thermodynamic regulation of the substrate/product couple in the acti
ve site of SCAD. Binding to the wild-type enzyme caused the midpoint potent
ial for the butyryl-CoA/crotonyl-CoA couple (E-BCoA/CCoA) to shift 14 mV ne
gative, stabilizing the oxidized product. Formation of product was found to
be even more favorable in complexes with the F160Y and F160W mutants, sugg
esting that the electrostatic environment around the flavin plays a role in
substrate/ product activation.