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https://hdl.handle.net/1959.11/13676
Title: | Conformational Dynamics of the Isoalloxazine in Substrate-Free ρ-Hydroxybenzoate Hydroxylase: Single-Molecule Studies | Contributor(s): | Brender, Jeffrey R (author); Dertouzos, Joe (author); Ballou, David P (author); Massey, Vincent (author); Palfey, Bruce A (author); Entsch, Barrie (author); Steel, Duncan G (author); Gafni, Ari (author) | Publication Date: | 2005 | DOI: | 10.1021/ja055171o | Handle Link: | https://hdl.handle.net/1959.11/13676 | Abstract: | ρ-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. | Publication Type: | Journal Article | Source of Publication: | Journal of the American Chemical Society, 127(51), p. 18171-18178 | Publisher: | American Chemical Society | Place of Publication: | United States of America | ISSN: | 1520-5126 0002-7863 1943-2984 |
Fields of Research (FoR) 2008: | 030403 Characterisation of Biological Macromolecules 060107 Enzymes |
Socio-Economic Objective (SEO) 2008: | 970106 Expanding Knowledge in the Biological Sciences 970103 Expanding Knowledge in the Chemical Sciences |
Peer Reviewed: | Yes | HERDC Category Description: | C1 Refereed Article in a Scholarly Journal |
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Appears in Collections: | Journal Article |
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