Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite

Abstract

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.

Characterization of the Bactericidal Effects of Sodium Nitroprusside and Other Pentacyanonitrosyl Complexes on the Food Spoilage BacteriumClostridium sporogenes

“…Carpenter et al (9) also reported the loss of activity of ferredoxin and pyruvate-ferredoxin oxidoreductase during treatment with nitrite. However, Payne et al (25,26) examined the effect of nitrite and iron-sulfur-nitrosyl complexes on ironsulfur clusters in Clostridium sporogenes cells (25) and on the isolated iron-sulfur proteins (26) and were unable to demonstrate a direct action of the compounds on the iron-sulfur proteins. A second possible effect is that of a general oxidizing agent.…”

“…Chemical determination of the sulfhydryl groups in cell fractions was used to assess the effect of SNP on the -SH content of each fraction. Electron paramagnetic resonance (EPR) spectroscopy of whole cells and cell fractions was used to observe the quantities of iron-sulfur proteins and their redox state in the cells (25). EPR signals were also detected from various species formed by reduction of the nitroprusside ion.…”

NO+, but not NO⋅, inhibits respiratory oscillations in ethanol‐grown chemostat cultures of Saccharomyces cerevisiae

“…The favoured chemistry of this highly reactive nitrosonium cation species is characterised by addition and substitution reactions with nucleophiles (electron‐rich bases and aromatics). At pH 7, nitrite is a rather ineffective nitrosylating agent (only 0.022% present as HNO 2 ); increased protonation (at pH 3.4 in the experiments described here) increase its effectiveness [23]. S ‐Nitrosothiols predominate over other reaction products under physiological conditions [24].…”

Nitrite inhibits hydrogen production and kills the cattle parasite Tritrichomonas foetus

“…This ‘phosphoroclastic’ system in C. sporogenes and C. botulinum is inhibited (Woods et al . 1981; Payne et al . 1990) and growth of the organisms is arrested (Duncan and Foster 1968) by concentrations in the mM range.…”

“…Thus, the oxidation of pyruvate to acetate and H 2 in both types of organisms requires pyruvate: ferredoxin oxidoreductase, ferredoxin and hydrogenase. This ÔphosphoroclasticÕ system in C. sporogenes and C. botulinum is inhibited (Woods et al 1981;Payne et al 1990) and growth of the organisms is arrested (Duncan and Foster 1968) by NO À 2 concentrations in the mM range. Interest in the cytotoxic effects of nitrosylating reagents and NO has become intense, as macrophages produce these agents to kill parasites (Fernandes and Assreuy 1997).…”