Conformational transitions accompanying oligomerization of yeast alcohol oxidase, a peroxisomal flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methan ol oxidation in methylotrophic yeasts. AO protein is synthesized in the cyt osol and subsequently sorted to peroxisomes where the active enzyme is form ed. To gain further insight in the molecular mechanisms involved in AO acti vation, we studied spectroscopically native AO from Hansenula polymorpha an d Pichia pastoris and three putative assembly intermediates. Fluorescence s tudies revealed that both Trp and FAD are suitable intramolecular markers o f the conformation and oligomeric state of AO. A direct relationship betwee n dissociation of AO octamers and increase in Trp fluorescence quantum yiel d and average fluorescence lifetime was found. The time-resolved fluorescen ce of the FAD cofactor showed a rapid decay component which reflects dynami c quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarka ble resemblance of pattern for purified AO and AO present in intact yeast c ells. Native AO contains a high content of ordered secondary structure whic h was reduced upon FAD-removal. Dissociation of octamers into monomers resu lted in a conversion of beta-sheets into alpha-helices. Our results are exp lained in relation to a 3D model of AO, which was built based on the crysta llographic data of the homologous enzyme glucose oxidase from Aspergillus n iger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.