ABSTRACT
An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.
Subject(s)
Enterococcus faecalis/enzymology , Multienzyme Complexes/isolation & purification , NADH, NADPH Oxidoreductases/isolation & purification , Water/biosynthesis , Chromatography, Affinity , Coenzymes , Electrons , Electrophoresis, Disc , Enterococcus faecalis/growth & development , Metals/analysis , Molecular Weight , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Spectrophotometry , Substrate Specificity , Triazines/pharmacologyABSTRACT
Conversion of estradiol and estrone to catechol estrogens by rat hypothalamic tissue but not by the cerebral cortex was demonstrated from the incubation of these tissues with estradiol-2-3H and estrone-2-3H and monitoring the incorporation of tritium into water. Direct evidence for this transformation was obtained by isolating the labelled phenazine derivative of 2-hydroxyestrone after hypothalamic incubation with estrone-4-14C.
