Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.

Retrospective data are presented for 6 adolescents ranging in age from 14 to 17 years, who were diagnosed with severe treatment-resistant major depression (TRD). Subjects were treated with one or more index courses of electroconvulsive therapy (ECT) followed by continuation ECT (C-ECT, up to 6 months of ECT) or maintenance ECT (M-ECT; ECT beyond 6 months) when necessary. Electroconvulsive therapy was continued until remission or until minimal residual symptoms were evident. Pharmacotherapy and psychotherapy were reintroduced during C-ECT or M-ECT. Premorbid functioning was achieved by 5 of 6 cases. Cognitive deficits were not evident. In fact, comparison of pre-ECT and post-ECT neuropsychological functioning revealed a trend toward improved auditory and verbal memory on most of the results. We concluded that C-ECT and M-ECT are useful and safe treatment strategies for selected adolescents with severe treatment-resistant depression, and symptom remission may be achieved without experiencing cognitive impairment.