NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly

• NADH oxidase and AhpC (Prx) form an oligomeric complex depending on ionic strength of ammonium sulfate.
• The complex formation is required for NADH oxidase–Prx system to rapidly reduce hydroperoxides.
• The solution structure of the complex was observed by SAXS analysis.

The NADH oxidase–peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from β-NADH passed through the secondary disulfide, Cys128–Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.