Topography of nitrate reductase of the cytoplasmic membrane of Escherichia coli: the nitrate-reducing site [proceedings].

Several aspects of the topography of the membrane-bound respiratory nitrate reductase (EC 1.7.99.4) of Escherichia coli have so far been explored (Kemp et al., 1975; Boxer & Clegg, 1975; Jones &Garland, 1977,1978; MacGregor &Christopher, 1978). Nevertheless, the location of the nitrate-binding site has not yet been positively identified. Garland et al. ( 1 9 7 5 ~ ) noted that the rate of passive entry of nitrate into spheroplasts as measured by osmotic swelling at relatively high concentrations of nitrate (25 mM) would, if extrapolated to lower concentrations of nitrate (0.25 mM), fail to support the known activities of nitrate reductase. This led to the suggestion that nitrate did not cross the cytoplasmic membrane before reduction. In retrospect this approach had two limitations: it would grossly underestimate the activity of a facilitated diffusion pathway (permease) with a low K , for nitrate, and it might fail to detect altogether a permease dependent on outward movement of nitrite in exchange for inward entry of nitrate. Furthermore, the assay conditions for measuring nitrate reductase activity in whole cells involve the use of Benzyl Viologen radical, which. because it is a hydrophobic permeant cation (Jones & Garland, 1977) could conceivably carry nitrate across the membrane as an uncharged complex. An alternative method of determining whether nitrate is reduced at the cytoplasmic or periplasmic face of the cytoplasmic membrane was suggested by analogy with the work of Palmieri & Klingenberg (1967) on the azide-sensitive site of mitochondrial cytochrome oxidase. Taking advantage of the fact that (i) azide is a weak acid with pK 3.34, (ii) hydrazoic acid diffuses across membranes very much more rapidly than does azide anion, and (iii) distribution of azide across a membrane will therefore be determined by any p H difference across the membrane, Palmieri & Klingenberg (1967) showed that pH gradients across the mitochondrial membrane which caused intramitochondrial accumulation of azide (i.e. matrix alkaline) also potentiated the inhibitory action of azide on cytochrome oxidase. So it was concluded that the azide-sensitive site was on the matrix face of the inner mitochondrial membrane. Azide is also an inhibitor of nitrate reductase, competing against nitrate with a K, of about 5pM (Forget, 1974). Garland et al. (1975a) used an indirect assay for nitrate reductase activity in cells of E. coli, and failed to find any effect of anticipated transmembrane pH gradients on the inhibitory effects of azide. This led to the suggestion that the nitrate-reducing site might