Resonance Raman spectroscopic evidence for an anionic flavin semiquinone in bovine liver monoamine oxidase.
The flavoprotein monoamine oxidase B (MAO B) from bovine liver, as isolated, has an unusual additional absorption band at 412 nm, which is similar to the absorption of its anionic flavin semiquinone form, (Fl.-), and other typical (Fl.-) flavoproteins. Denaturation of the enzyme results in the elimination of this anomalous absorption. The resonance Raman (RR) spectrum of MAO B as isolated is virtually identical to that of its dithionite-reduced (Fl.-) form. Both spectra show features similar to those of the RR spectrum of the (Fl.-) form of Aspergillus niger glucose oxidase (GO) in the region between 300 and 1700 cm-1 with 406.7 nm excitation. These features are readily distinguishable from those of oxidized flavin, neutral flavin semiquinone, and hemoprotein, strongly suggesting the presence of an (Fl.-) form in MAO B as isolated, even with preparations isolated in the absence of light. There are significant differences between the RR spectra of the (Fl.-) form of MAO B and those of GO or the published RR spectra of the (Fl.-) form of D-amino acid oxidase with excess substrate analog. At least some of these differences can be attributed to the different binding of flavin in the three enzymes. No EPR signals due to (Fl.-) are observed in MAO B as isolated. The dithionite-reduced (Fl.-) form exhibits approximately 50% less EPR signal than that expected from the absorption spectrum, which suggests a possible coupling of the (Fl.-) flavin with a paramagnetic center of unknown identity in the protein. The implications of these observations on MAO B with the current view of its catalytic mechanism are discussed.
Yue
K T
KT
Department of Physics, Emory University, Atlanta, Georgia 30322.
Bhattacharyya
A K
AK
Zhelyaskov
V R
VR
Edmondson
D E
DE
D000818:Animals; D002417:Cattle; D004227:Dithionite; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000032:analysis; D005420:Flavoproteins / Q000737:chemistry* / Q000302:isolation & purification; D019791:Guanidine; D006146:Guanidines; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D008930:Mitochondria, Liver / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman / Q000379:methods
D005420:Flavoproteins; D006146:Guanidines; D007527:Isoenzymes; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; C029276:flavin semiquinone; D008995:Monoamine Oxidase; D019791:Guanidine