Isolation of ferredoxin-nicotinamide-adenine dinucleotide phosphate (oxidized) reductase from a prokaryote [proceedings].

The flavoprotein NADPH-ferredoxin oxidoreductase (EC 1.6.99.4) is a component of the photosynthetic electron-transport chain, catalysing the reduction of NADP+ with reduced ferredoxin (Shin et al., 1965). The enzyme also possesses activity as a NADPH diaphorase (Avron & Jagendorf, 1956), a transhydrogenase (Keister et a/., 1960) and NADPH-cytochrome f reductase (Zanetti & Forti, 1966) all three activities being independently reported before its physiological role in photosynthetic electron transport was recognized. Ferredoxin-NADP+ reductase was first purified from Spinacea oleracea (spinach) by Shin et al. (1963). The enzyme has also been purified to varying degrees from Pinus pinea (pine) by Firenzuoli et al. (1968), from Tsuga canadensis (eastern hemlock) by Riov & Brown (1976), and from Anabaena variabilis (Susor & Krogmann, 1966) and Anabaena cylindrica (Codd et al., 1974). The spinach enzyme possesses 1 mol of FAD per mol of protein (Keirns & Wang, 1972) and forms a 1 : 1 complex with ferredoxin (Shin & San Pietro, 1968). However, the molecular weight of the plant enzyme remains in doubt (Table 1). We now wish to report the isolation and some properties of the ferredoxin-NADP+ reductase from a prokaryote, the filamentous non-heterocystous cyanobacterium Nostoc strain MAC. Nostoc strain MAC was grown autotrophically in the light as described in Hutson etal. (1978).Theyieldfromeight 10-litreflaskswas typically 140gofcellpaste, which was resuspended in 200ml of 0.01 M-Tris/HCl, pH7.8. Cell suspensions were added slowly with vigorous stirring to 2.0 litres of acetone at -20°C. This and all subsequent stages of the purification were carried out in a cold room at 4"C, using buffers of pH7.8 at this temperature unless otherwise stated. Acetone-dried powder from two batches (approx. 5Og) was homogenized in 300ml of O.lSM-Tris/HCl and the suspension stirred for approx. 15h after addition of 5mg each of deoxyribonuclease and ribonuclease to degrade polynucleotides. The mixture was centrifuged at 40000g for 60min and the supernatant was adjusted to 30 % saturation with (NH4)2S04. Precipitated protein was removed by centrifugation at 40000g for 30min and the supernatant adjusted to 60% saturation with (NH4)2S04. The precipitate obtained on a further centrifugation was dissolved in 200ml of O.OSM-Tris/HCI and dialysed for 24h against 5 litres of 0 . 0 2 ~ -