CHANGES IN THE CATALYTIC PROPERTIES OF P-HYDROXYBENZOATE HYDROXYLASE CAUSED BY THE MUTATION ASN300ASP

By site-directed mutagenesis, we have changed Asn 300 to Asp in p-hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) from Pseudomonas aeruginosa. In the wild-type (WT) enzyme, residue 300 is in contact with the isoalloxazine ring of the active-site FAD; in the Asn300Asp mutant, this side chain has moved by about 5 Angstrom, altering the protein structure [Lah, M. S., Palfey, B. A., Schreuder, H. A., and Ludwig, M. L. (1994) Biochemistry (following paper in this issue)]. The structural changes are responsible for profound catalytic and dynamic effects. The flavin of PHBH is reduced by NADPH in the first half of catalysis. The mutation has decreased this fate 330-fold, apparently by affecting the reactive orientation of the isoalloxazine and pyridine rings. Furthermore, the redox potential of the flavin is lower in the mutant enzyme than in WT by 20-40 mV. The reduced flavin of PHBH reacts with O-2 to form a flavin C(4a)-hydroperoxide, which is the species that transfers oxygen to the aromatic substrate. Previous studies indicated that the enzyme promotes the hydroxylation reaction in part by activating the substrate through lowering the phenolic pK(a). The Asn300Asp mutant does not lower the substrate pK(a). As a consequence of this, and also an enhanced stability of the flavin C(4a)-hydroperoxide the hydroxylation is 50-fold slower in the mutant than in WT. However, despite the slow rate of the hydroxylation reaction, no H2O2 is formed by the competitive elimination reaction. The kinetic stability of the flavin C(4a)-hydroxide formed by the hydroxylation was also enhanced by the mutation. By studying the effects of the inhibitor azide on the oxidative sequence, we were able to conclude that the inhibitory site is readily accessible to solvent; azide binding at a second site slowly displaces the substrate from the reduced enzyme. The mutation has profoundly slowed the rates of ligand binding to the enzyme. Kinetic studies of binding indicated the presence of several enzyme conformations. Thus, the mutation of this one residue interferes with the orientation of pyridine nucleotide and flavin during reduction, stabilizes flavin C(4a) intermediates, prevents substrate ionization, and alters the rates and strengths of ligand binding.