Conformational Dynamics of the Isoalloxazine in Substrate-Free p-Hydroxybenzoate Hydroxylase: Single-Molecule Studies Export

@article{citeulike:4305994, 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.}, author = {Brender, Jeffrey R. and Dertouzos, Joe and Ballou, David P. and Massey, Vincent and Palfey, Bruce A. and Entsch, Barrie and Steel, Duncan G. and Gafni, Ari}, citeulike-article-id = {4305994}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/ja055171o}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/ja055171o}, doi = {10.1021/ja055171o}, journal = {Journal of the American Chemical Society}, keywords = {fad, kinetics, phbh, single-molecule}, month = {December}, number = {51}, pages = {18171--18178}, posted-at = {2009-04-14 05:52:41}, priority = {4}, title = {Conformational Dynamics of the Isoalloxazine in Substrate-Free p-Hydroxybenzoate Hydroxylase: Single-Molecule Studies}, url = {http://dx.doi.org/10.1021/ja055171o}, volume = {127}, year = {2005} }

TY - JOUR ID - citeulike:4305994 L3 - citeulike-article-id:4305994 N2 - 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. IS - 51 JF - Journal of the American Chemical Society EP - 18178 TI - Conformational Dynamics of the Isoalloxazine in Substrate-Free p-Hydroxybenzoate Hydroxylase: Single-Molecule Studies VL - 127 SP - 18171 KW - fad KW - kinetics KW - phbh KW - single-molecule AU - Brender, Jeffrey AU - Dertouzos, Joe AU - Ballou, David AU - Massey, Vincent AU - Palfey, Bruce AU - Entsch, Barrie AU - Steel, Duncan AU - Gafni, Ari PY - 2005/12/01/ UR - http://dx.doi.org/10.1021/ja055171o ER -