Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E-coli:Direct evidence that VHb enhances recombinant protein production

Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (V Hb) typically produce more protein than unengineered cells, and it has gene rally been assumed that VHb is responsible for this effect. Here, using mat ched strains of E. coli that bear a recombinant alpha -amylase gene (MK57) or the alpha -amylase gene and vgb (MK79), we provide evidence supporting t his assumption. Sodium nitrite (which is known to inhibit heme proteins) wa s tested over a range of concentrations regarding effects on growth, alpha -amylase production, respiration, and VHb function in MK57 and MK79. Nitrit e concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains , while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhi bit VHb while having a much smaller effect on cytochrome oxidase. Direct me asurements of VHb showed, in fact, that the same nitrite concentrations gre atly decreased the levels of active (ferrous) and, to a somewhat lesser ext ent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite c oncentrations reversed the advantage regarding alpha -amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb wi th the increase in alpha -amylase production.