Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis

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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 metalfree and has a molecular mass of about 51000 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 × 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.