Fluorescence studies on dopamine beta-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+.
The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology*; D004299:Dopamine beta-Hydroxylase; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D010618:Phenanthrolines / Q000494:pharmacology; D011193:Potassium Iodide; D012492:Salts; D013050:Spectrometry, Fluorescence
D002614:Chelating Agents; D010618:Phenanthrolines; D012492:Salts; D011193:Potassium Iodide; C028559:bathocuproine sulfonate; D003300:Copper; D004299:Dopamine beta-Hydroxylase