Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase.

Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.