Determination of the oligomeric states of human and rat monoamine oxidases in the outer mitochondrial membrane and octyl beta-D-glucopyranoside micelles using pulsed dipolar electron spin resonance spectroscopy.
Human monoamine oxidase A (hMAOA) is considered to be unique among mammalian MAOs in having a non-conservative Glu-X-Lys mutation (X being 151 in MAOAs and 142 in MAOB's), which is suggested to be the reason for its monomeric structure. This hypothesis has been tested in this work. A pargyline based nitroxide spin labeled irreversible inhibitor (ParSL) was used as a MAO active site specific spin probe to measure intersubunit distances in detergent (octyl beta-d-glucopyranoside, OGP) purified and OMM bound forms by a pulsed dipolar ESR spectroscopic (PDS) technique. In a parallel approach, the covalent flavin cofactor present in the MAO active sites was reduced to its respective anionic flavin semiquinone and used for measuring inter-flavin distances in detergent purified samples. The measured interspin distances are within 0.1-0.3 nm of those estimated from the available dimeric crystal structures of human MAOB and rat MAOA and show that all human and rat MAOs exist as dimers in the OMM. In the OGP micelle, however, human and rat MAOAs exist only partially (<or=50%) as dimers, whereas human and rat MAOBs exist entirely as dimers. The Lys-151-Glu mutant of human MAOA and the Glu-142-Lys mutant of human MAOB exhibit similar spectral properties as the corresponding wild-type enzymes. Therefore, no role of the Glu-X residue in stabilizing dimeric structures of MAOs was found. The monomeric crystal structure reported for human MAOA is thus a result of its instability in the OGP micelles. The general applicability of the PDS technique to structural studies of membrane proteins in their native membrane environments and detergent purified forms is discussed.
Upadhyay
Anup K
AK
Department of Biochemistry, Emory University, 1510 Clifton Road, Atlanta, Georgia 30322, USA.
Borbat
Peter P
PP
Wang
Jin
J
Freed
Jack H
JH
Edmondson
Dale E
DE
D000818:Animals; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005960:Glucosides / Q000737:chemistry*; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008823:Micelles; D008928:Mitochondria / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000737:chemistry*; D051381:Rats
D001704:Biopolymers; D005960:Glucosides; D008823:Micelles; C018619:octyl-beta-D-glucoside; D008995:Monoamine Oxidase