Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence.
Time-resolved fluorescence studies were carried out on the FAD bound to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the 50-500-ps time region and a longer one in the range 1.5-3.5 ns. The shorter-lifetime component has a larger contribution in the presence of substrate (p-hydroxybenzoate) or inhibitor (p-aminobenzoate). The quenching of the fluorescence is both static and dynamic in nature. The decay of fluorescence anisotropy shows that the FAD environment is both flexible and rigid. The FAD mobility can be enhanced by dilution of the enzyme, by raising the temperature, or by the binding of substrate or inhibitors. The anisotropy results are interpreted in part in terms of a monomer-dimer equilibrium, whereby the FAD in the monomer contains much more flexibility. The above-mentioned effects induce a shift of the equilibrium to the monomeric side. From a constrained parameter fitting the dissociation constant is estimated to be about 1 microM for the free enzyme and somewhat higher for the binary complexes between the enzyme and substrate or inhibitor. pH variation has only a slight effect on fluorescence or anisotropy decay parameters, while dimethylsulfoxide appears to promote dissociation into monomers by weakening hydrophobic interaction between the subunits. The results are discussed in the light of newly developed insights into the functional role of rapid structural fluctuations in enzyme catalysis.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase