Chelator‐induced inhibition of copper metalloenzymes in denitrifying bacteria

Abstract

Copper (Cu) is required by the enzyme nitrous oxide reductase (N 2 OR), which catalyzes the last step of the complete denitrification pathway in denitrifying bacteria. Some denitrifiers also require copper for nitrite reductase (NiRK), whereas others use the iron nitrite reductase (NiRS). We report the inhibition of the activity of these enzymes in three strains of denitrifiers (two containing NiRK, the other NiRS), by forming nonbioavailable complexes with 1,4,8,11‐tetraazacyclotetradecane1,4,8,11‐tetraacetic acid hydrochoride hydrate (TETA), a strong Cu(II) chelator, and tetrathiomolybdate (TTMo), a strong Cu(I) chelator. Both ligands complex Cu with stability constants comparable to naturally occurring ligands and much more strongly than other widely used chelators, such as ethylenediaminetetraacetic acid. Addition of TETA to growth media lowered free Cu 2+ concentrations below 10 −16 mol L −1 and induced Cu limitation in all organisms. While Cu is strongly complexed in seawater, 10 −16 mol L −1 free Cu 2+ is lower than most reported values, suggesting that the organisms have evolved high‐affinity Cu transport systems. TTMo had different effects, and inhibited NiRK more effectively than N 2 OR. It is likely that TTMo inhibits NiRK through direct, noncompetitive inhibition, as reported for other reduced sulfur compounds, rather than by inducing Cu limitation. The activity of NiRK may be sensitive to trace levels of reduced sulfur, which could account for its scarcity in marine systems. While Cu limitation of denitrification is probably uncommon in aquatic systems, the presence of reduced sulfur compounds may induce Cu limitation or enzyme inhibition leading to the accumulation of nitrite and nitrous oxide.