A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in Amphibacillus xylanus

Abstract

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with ␤-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide ( We have reported previously on a new group of facultatively anaerobic bacteria isolated from alkaline compost (17). Since the bacteria have unique phenotypic and chemotaxonomic characteristics (18), as well as bioenergetic properties (13), we named one of the isolate Amphibacillus xylanus (18). A. xylanus, which lacks a respiratory system and heme proteins, including catalase, grows well and has the same growth rate and cell yield under strictly anaerobic and aerobic conditions (18). This is due to the presence of anaerobic and aerobic pathways producing similar amounts of ATP (19). We purified NADH oxidase from aerobically grown A. xylanus (20) and found that hydrogen peroxide is formed during the reaction of the NADH oxidase. We have also found that the enzyme reduces hydrogen peroxide in the presence of additional free flavin adenine dinucleotide (FAD); this may be of physiological relevance because of the presence of 13 M free FAD in aerobically growing cells. Such behavior indicates that the A. xylanus NADH oxidase has unique functional properties that are different from those of other known NADH oxidases (8,10,12,27,(31)(32)(33).The amino acid sequence of A. xylanus NADH oxidase exhibits up to 51.2% identity to the alkyl hydroperoxide reductase F-52a flavoprotein component (AhpF) from Salmonella enterica (36), but the ability of the latter to reduce hydrogen peroxide was not reported (11). We have shown that both enzymes reduce not only alkyl hydroperoxides but also hydrogen peroxide in the presence of the 22-kDa protein component (AhpC) of the alkyl hydroperoxide reductase from Salmonella enterica, and the turnover numbers of the reactions catalyzed by the NADH oxidase are extremely high compared with those of known peroxide-reducing enzymes (21). Complete reduction of the NADH oxidase by dithionite required 6 electron equivalents/mol of enzyme-bound flavin (25,26). Such behavior indicated the presence of redox centers in addition to the FAD, and these were shown to be two disulfides, Cys128-Cys131 and Cys337-Cys340. To provide information about the relationship between such high turnover numbers and the three redox centers of the enzyme, the reductive half-reactions of the wild-type and the mutant (C337S/C340S) NADH oxidase were investigated and have been reported in a previous publication (22).Hydrogen peroxide is presumed to be accumulated in aerobically growing cells of A. xylanus, which lack catalase and a respiratory chain (18), because the NADH oxidase should regenerate NAD ϩ from NADH formed in the aerobic pathway and produce hydrogen peroxide from oxygen. The ability to get rid of hydrogen peroxide must be high for the cells to be able to survive under aerobic conditions. In the presence of free FAD or the AhpC protein, the NADH oxidase shows scavenging activity for hydrogen peroxide (21,2...

Generating disulfides enzymatically: Reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p

“…Precedents for reduction of exogenous flavins by flavoproteins exist, including the reductase modules of two-component monooxygenases (reviewed in ref. 27) and an Amphibacillus xylanus NADH oxidase (28). Although the capacity for electron transfer between flavoproteins and exogenous flavins in vitro appears to be a general phenomenon (24), its physiological implications in the present case are less clear.…”

Molecular cloning and transcriptional analysis of the alkyl hydroperoxide reductase genes from Pseudomonas putida KT2442.

“…Comparison of P. putida AhpC with corresponding protein from S. typhimurium , Xanthomonas campestris pv. phaseoli (Loprasert et al, 1997), P. aeruginosa (Stover et al, 2000) and Amphibacillus xylanus (Niimura et al, 2000) showed 69, 66, 65, and 65% overall similarity, respectively (data not shown). Two highly conserved cysteine residues among bacterial AhpCs are found at 47 and 166.…”

Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria

“…The enzymes catalyze the reduction of oxygen by NADH to form hydrogen peroxide. However, in the presence of a 21-kDa disulfide-containing redox protein (AhpC), now commonly referred to as peroxiredoxin (Prx), the NADH oxidases also showed extremely high reductase activity for both hydrogen peroxide and alkyl hydroperoxides (26,(28)(29)(30). These NADH oxidases thus belong to a growing new family of peroxiredoxin oxidoreductases (PrxR) (38) and are involved not only in the regeneration of NAD but also in the removal of peroxides.…”