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Time-resolved fluorescence studies were carried out on the FAD bound top-hydroxybenzoate hydroxylase from Pseudomonas Juorescens. The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the SO -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 pM 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.The enzyme p-hydroxybenzoate hydroxylase from Pseudomonas Jluorescens belongs to the class of flavindependent monooxygenases. It catalyzes the conversion of p-hydroxybenzoate into 3,4-dihydroxybenzoate. The enzymatic mechanism has been studied in detail [I]. It has been shown that the enzymatic reaction is strictly dependent on NADPH [2]. However, recently we observed that NADH can also serve as an electron donor in the catalytic reaction, though in a less efficient way than NADPH (Van Berkel and Muller, unpublished results). The substrate and some inhibitors also function as effectors, i.e. accelerate the rate of reduction of the protein-bound FAD [3]. In the past few years the chemical and physical properties of the enzyme have been investigated by various techniques. A low-resolution (0.25-nm) threedimensional model is available [4] and the entire sequence of the enzyme is known [5, 61. In addition it has been shown that the enzyme exhibits a complex quaternary structure [7]. In solution the enzyme is present as monomer, dimer and higher order structures [7]. Very recently we succeeded in separating pure dimeric enzyme from the mixture by column chromatography (Van Berkel and Muller, unpublished results).The possibility now to obtain a well defined enzyme preparation and the fact that the enzyme-bound FAD shows a relatively large fluorescence quantum yield, lead us to inAbbreviations. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Hepps, 4-(2-hydroxyethyl)-l-piperazinepropanesulfonic a...