Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis.
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.
Schmidt
H L
HL
Stöcklein
W
W
Danzer
J
J
Kirch
P
P
Limbach
B
B
D002846:Chromatography, Affinity; D003067:Coenzymes; D004583:Electrons; D004589:Electrophoresis, Disc; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000254:growth & development; D008670:Metals / Q000032:analysis; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity; D014227:Triazines / Q000494:pharmacology; D014867:Water / Q000096:biosynthesis*
D003067:Coenzymes; D008670:Metals; D009097:Multienzyme Complexes; D014227:Triazines; D014867:Water; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases