The purification of a cd1-type nitrite reductase from, and the absence of a copper-type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor

Abstract

Thiosphaera pantotropha has been reported to contain a copper-type nitrite reductase on the basis that the copper chelator diethyldithiocarbamate inhibited the overall process of denitrification. It is now shown that nitrous oxide reduction is 100% inhibited by 10 mM diethyldithiocarbamate or 100 microM azide. We also found that both these inhibitors partially inhibited nitrite reduction in this organism. We purified the nitrite reductase of T. pantotropha and found that it was of the cytochrome cd1 type, contrary to the published report of it being a copper-type nitrite reductase. This is of importance since T. pantotropha is capable of aerobic nitrite reduction. The only detectable nitrite reductase in anaerobically or aerobically grown cells is the cd1 type. We also purified a small copper-containing protein, pseudoazurin. Pseudoazurin was found to be capable of donating electrons to the cd1-type nitrite reductase in vitro, and its copper centre was chelated by diethyldithiocarbamate. Since nitrite reduction is partially inhibited by diethyldithiocarbamate, it is thought that pseudoazurin is an electron donor to nitrite reductase in vivo.