Purification and characterisation of NADH oxidase from Thermus aquaticus YT-1 and evidence that it functions in a peroxide-reduction system.
A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT-1 by chromatography on DEAE-cellulose and AMP-Sepharose. The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit. The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH. The blue flavin semiquinone is stabilised during photo-chemical reduction of the enzyme. Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases. Reduction by the substrate NADH is incomplete, with the formation of a new long-wavelength absorption underlying the semiquinone absorption. Amino acid sequencing showed that the T aquaticus enzyme resembles other microbial flavoenzymes that function in two-enzyme systems for the reduction of peroxides, and which contain two redox-active disulphide groups in addition to the flavin. The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6-dichloroindophenol, and 5,5'dithiobis(2,2'-dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide-reducing system of Salmonella typhimurium. The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T. aquaticus NADH oxidase functions by mediating electron transfer from enzyme-bound reduced flavin to O2. The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor.
Toomey
D
D
Department of Biochemistry, University College Dublin, Belfield, Ireland.
Mayhew
S G
SG
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology* / Q000254:growth & development
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases