Nonsymbiotic hemoglobins are broadly present across evolution; however, the function of these proteins is unknown. Cultured maize cells have been transformed to constitutively express a barley 
hemoglobin gene in either the sense (HB+) or antisense (HB−) orientation. 
Hemoglobin protein in the 
transformed cell lines was correspondingly higher or lower than in 
wild type cells under normal atmospheric conditions. Limiting 
oxygen availability, by placing the cells in a 
nitrogen atmosphere for 12 hours, had little effect on the energy status of cells constitutively expressing 
hemoglobin, but had a pronounced effect on both 
wild type and HB− cells, where ATP levels declined by 27% and 61% respectively. 
Energy charge was relatively unaffected by the treatment in HB+ and 
wild type cells, but was reduced from 0.91 to 0.73 in HB− cells suggesting that the latter were incapable of maintaining their energy status under the 
low oxygen regime. Similar results were observed with P. aeruginosa cells transformed with an Hb 
expression vector. It is suggested that nonsymbiotic hemoglobins act to maintain the energy status of cells in 
low oxygen environments and that they accomplish this effect by promoting glycolytic flux through NADH oxidation, resulting in increased substrate level 
phosphorylation. Nonsymbiotic hemoglobins are likely ancestors of an early form of 
hemoglobin that sequestered 
oxygen in 
low oxygen environments, providing a source of 
oxygen to oxidize NADH to provide ATP for 
cell growth and development. This in turn suggests that cells containing increased levels of Hb 
protein will survive longer under low 
oxygen tension or 
high energy demand.