The phosphoroclastic system was demonstrated in cell-free extracts of clostridium sporogenes by the production of carbon dioxide, acetyl phosphate, ATP and reduced NAD in the presence of pyruvate. The kinetics of acetyl phosphate production and NAD reduction were investigated. The addition of sodium nitrite to a suspension of C. sporogenes in glucose medium resulted in a rapid decrease in intracellular ATP concentration which was accompanied by an accumulation of pyruvate in the medium. This accumulation of pyruvate was caused by inhibition of phosphoroclastic system by nitrate. Nitrite inhibits this system by reaction of nitric oxide, formed from nitrate, with the non-haem iron of pyruvate:ferredoxin oxidoreductase.

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The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the ironsulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sprogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitrite with 10 mM-ascorbate stopped growth completely. In partially-purified preparations 4.1 mM-NaNO, and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78 %) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO),(SR),] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxh were shown by EPR spectroscopy to be resistant to treatment with 3.6 mM-NaNO, and 3.6 nml-ascorbate. It is concluded that the eKects of nitrite on pre-formed iron-snlphur proteins are not convincing as a basis for the lethal effects on bacterial cells.

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