flavin-adenine-dinucleotide and 2-4-dichlorophenol

Cellobiose oxidase (CBO) from Phanerochaete chrysosporium can utilize dichlorphenol-indophenol (Cl2Ind) and cytochrome c as effective electron acceptors for the oxidation of cellobiose. However, the pH dependencies of activity for these electron acceptors are significantly different. Both compounds act as effective electron acceptors at pH 4.2, whereas only dichlorophenol-indophenol is active at pH 5.9. To explain this discrepancy, the pH dependencies of the reduction rates of FAD and heme, respectively, in CBO by cellobiose have been investigated by stopped-flow spectrophotometry. Both FAD and heme are reduced with a high rate constant at pH 4.2. In contrast, at pH 5.9, only FAD reduction is fast, while the reduction of the heme is extremely slow. As a conclusion, the reduction of cytochrome c by CBO is dependent on heme, which functions at a lower pH range compared to reduction of FAD.

Topics: Carbohydrate Dehydrogenases; Chlorophenols; Cytochrome c Group; Flavin-Adenine Dinucleotide; Heme; Hydrogen-Ion Concentration; Indophenol; Kinetics; Oxidation-Reduction; Spectrophotometry; Sporothrix

1. 2,4-Dichlorophenol hydroxylase has been purified 13-fold from Acinetobacter grown on 2,4-dichlorophenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80-90% pure by electrophoresis. 2. The enzyme has a relative molecular mass of about 240 000 and consists of four subunits of identical size. 3. The enzyme contains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme. 4. The reaction catalysed is an NADPH-dependent hydroxylation of 2,4-dichlorophenol with the formation of 3,5-dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH. 5. The enzyme possesses broad effector specificity. In addition to 2,4-dichlorophenol, 4-chlorophenol and 4-chloro-2-methylphenol are true substrates for the enzyme. A number of 'non-substrate effectors' has been found. 6. The enzyme is sensitive to thiol-inhibiting reagents.

Topics: Acinetobacter; Chlorophenols; Drug Stability; Flavin-Adenine Dinucleotide; Hydroxylation; Kinetics; Macromolecular Substances; Mixed Function Oxygenases; Molecular Weight; NADP; Substrate Specificity