GENERATION OF REDUCING POWER IN CHEMOSYNTHESIS .V. MECHANISM OF PYRIDINE NUCLEOTIDE REDUCTION BY NITREIT IN CHEMOAUTOTROPH NITROBACTER AGILIS

1. Cell-free extracts from Nitrobacter catalyzed an energy-dependent reduction of NAD+ by NO2-; the process could be driven either at the expense of added ATP or by the energy generated from NO2- oxidation. 2. The reduction of cytochrome c by NO2- was observed to be an energydependent reaction which involved reversal of electron transfer from cytochrome a1. The subsequent energy-linked reduction of the flavoproteins and pyridine nucleotides occurred concomitantly with the oxidation of cytochrome c. 3. The reduction of each mole of NAD+ by NO2- required the utilization of approx. 5 moles of ATP. These observations are in harmony with the calculated energetics of the overall reverse electron flow process which involves a free energy gap of some 35 kcal. 4. Under conditions when ATP was limiting, the energy-linked reduction of 1 molecule of NAD+ by nitrite required the concomitant oxidation of 2 molecules of cytochrome c. However, when the process was driven by an optimal ATP concentration (0.7 mM), the rate of NAD+ reduction was about 1.5-fold compared to the rate of cytochrome c oxidation. 5. The process of energy-linked reversal of electron transfer in Nitrobacter was markedly sensitive to the inhibitors of the flavoprotein systems as well as to antimycin A or 2-n-heptyl-4-hydroxyquinoline N-oxide. In addition, CN- was observed to be a potent inhibitor. The uncouplers of oxidative phosphorylation caused a strong inhibition of the ATP-linked reverse electron transfer, and the energy transfer from ATP to drive reverse electron flow was also inhibited by oligomycin. © 1969.