CRYSTAL STRUCTURE OF CYTOCHROME c552 FROM Pseudomonas nautica
Kieron Brown1, Didier Nurizzo1, Stephane Besson2, Jose Moura2, Mariella Tegoni1, Christian Cambillau1
1AFMB-CNRS, 31 chemin Joseph Aigiuer,
13402 Marseille, France
2Centro de Quemica eiotecnologia,
Universidade Nova de Lisboa, Portugal
The purification and characterisation of enzymes and electron carriers involved in the so-called respiratory pathway of marine denitrifiers is the first step in the study of the biochemistry of denitrification in marine ecosystems. An understanding of the physio-logical and biochemical aspects of denitrification in marine environments is important, since nitrates may be viewed both as pollutants and as fertilisers.
Cytochrome c552 is believed to be the electron donor to the nitrite reductase. Nitrite reductases perform two roles: the reduction of nitrite to nitric oxide
NO-2 + 2 H+ ---> NO + H2O
and the catalysis of the reduction of molecular oxygen to water
O2 + 4H+ + 4e- ---> 2H2O
A c-type monohemic ferricytochrome c552 was isolated from the soluble extract of the marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions and purified in three stages (anionic exchange/cationic exchange/gel fitration). Crystals were obtained from c552 at an initial concentration of 12.4 mg/ml using 1.7M ammonium sulphate and 0.1M Na-citrate at pH5.6
Cytochrome c552 crystallises in the space group P3112 with cell dimensions a = b = 121.9 A, c = 139.9 A, a = b = 90o g = 120o, with 8 molecules per asymmetric unit. The Matthews number of 5.4A3/Da corresponds to a solvent content of 68%.
MAD data were collected at 110K on DW21b at LURE at wavelengths deduced from the XANES scan of the Fe K-edge. Eight sites were identified from the dispersive Patterson using SHELX, and MAD phasing to a resolution of 2.85 A carried out by MLPHARE, giving a mean figure of merit of 0.44. The correct hand was identified by examination of the relevent electron density maps using the raw MAD phases. Building of the C-alpha main chain was carried out using option TPPR in TURBO, and preliminary refinement performed by XPLOR.