title: Conformational dynamics of the isoalloxazine in substrate-free p-hydroxybenzoate hydroxylase: single-molecule studies.
abstract: p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric enzyme in which each subunit noncovalently binds one molecule of FAD in the active site. PHBH is a model system for how flavoenzymes regulate reactions with oxygen. We report single-molecule fluorescence studies of PHBH in the absence of substrate that provide data consistent with the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an "in" conformation and a more exposed or "open" conformation. The isoalloxazine is observed to move between these conformations in the absence of substrate. Studies with the Y222A mutant form of PHBH suggest that the exposed conformation is fluorescent while the in-conformation is quenched. Finally, we note that many of the single-molecule-fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to hypothesize a model in which one flavin in the dimer inhibits the motion of the other.
authors.affiliation: Biophysics Research Division, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Brender
authors.forename: Joe
authors.initials: J
authors.lastname: Dertouzos
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Vincent
authors.initials: V
authors.lastname: Massey
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Palfey
authors.forename: Barrie
authors.initials: B
authors.lastname: Entsch
authors.forename: Duncan G
authors.initials: DG
authors.lastname: Steel
authors.forename: Ari
authors.initials: A
authors.lastname: Gafni
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D005182:Flavin-Adenine Dinucleotide; D015109:4-Hydroxybenzoate-3-Monooxygenase