AMPHIBACILLUS XYLANUS NADH OXIDASE AND SALMONELLA-TYPHIMURIUM ALKYL-HYDROPEROXIDE REDUCTASE FLAVOPROTEIN COMPONENTS SHOW EXTREMELY HIGH SCAVENGING ACTIVITY FOR BOTH ALKYL HYDROPEROXIDE AND HYDROGEN-PEROXIDE IN THE PRESENCE OF SALMONELLA-TYPHIMURIUM ALKYL-HYDROPEROXIDE REDUCTASE 22-KDA PROTEIN-COMPONENT

The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide, The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase hom Salmonella typhimurium, AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of tbe Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide. Both enzymes' K-m values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately, V-max values for hydrogen peroxide or cumene hydroperoxide reduction are >10,000 min(-1) at 25 degrees C, These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C340S. Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S). Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.