Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins.
Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.
Wille
Georg
G
Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany. georg@bc.biochemtech.uni-halle.de
Ritter
Michaela
M
Friedemann
Rudolf
R
Mäntele
Werner
W
Hübner
Gerhard
G
D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011771:Pyruvate Oxidase / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013552:Swine; D014867:Water / Q000737:chemistry
D005420:Flavoproteins; D012996:Solutions; D014867:Water; D005182:Flavin-Adenine Dinucleotide; D005949:Glucose Oxidase; D011771:Pyruvate Oxidase; D003605:D-Amino-Acid Oxidase