Sulphite- and NADH-dependent Nitrate Reductase from Thiobacillus denitrificans

Abstract

SUMMARY: A membrane-bound nitrate reductase from Thiobacillus denitrificans can utilize either sulphite or NADH as an electron donor. The sulphite-dependent nitrate reductase activity was released from the membrane by treatment with sodium deoxycholate. Cytochrome c and FAD were separated from the solubilized enzyme by heat treatment and subsequent chromatography on DEAE-cellulose. The bacterial cytochrome c and a preparation of horse-heart cytochrome c served as electron mediators to the solubilized sulphite-dependent nitrate reductase activity with apparent K m values of 1·5 and 1·3 μ m respectively. The NADH-linked enzymic activity, which was unstable during storage, was re-activated with reduced glutathione. It was also inactivated after treatment with deoxycholate but this effect was reversed by menadione. A possible scheme for electron transport for the sulphite- and NADH-dependent enzyme is proposed.