A newly developed on-line P-31 NMR system has been used to investigate the effect of Vitreoscilla hemoglobin (VHb) expression on the steady-state level of ATP in Escherichia coli under oxygen-limited conditions. The VHb-expressing strain GRO21 exhibited a higher growth rate on a minimal fed-batch medium after the onset of oxygen limitation and maintained the same level of ATP pool and transmembrane Delta pH as the slower growing wild-type strain MG1655, indicating that the net ATP accumulation rate (the rate at which ATP increases per unit volume of culture per unit time) was enhanced in the VHb-expressing strain. Further direct evidence of the enhanced flux of P-i-ATP (catalyzed by ATPase) in GRO21 was obtained in a saturation transfer study on respiring cells. VHb-expressing cells respiring on succinate exhibited a P-i-ATP flux 30 +/- 5% higher than that of an otherwise isogenic strain that did not express VHb.