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

Citation
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
Citations number
51
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
40
Issue
25
Year of publication
2001
Pages
7720 - 7728
Database
ISI
SICI code
0006-2960(20010626)40:25<7720:ROASII>2.0.ZU;2-B
Abstract
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.