CONFORMATIONAL CHANGES OCCURRING UPON REDUCTION IN NITRITE REDUCTASE FROM Pseudomonas aeruginosa.

Didier Nurizzo1, Francesca Cutruzzola2, Marzia Arese2, Dominique Bourgeois3, Maurizio Brunori2, Christian Cambillau1 and Mariella Tegoni1

1Architecture et Fonction des Macromolécules Biologiques, U.P.R.9039-C.N.R.S., I.B.S.M., 31, Ch.Joseph Aiguier, Marseille Cedex 20 (France)
2 Dipartimento di Scienze Biochimiche, Universita di Roma "La Sapienza", P. le Aldo Moro 5, 00185 Roma (Italy)
3 E.S.R.F., BP 220, 38043 Grenoble, (France)

Keywords: NO, nitrite reductase, Pseudomonas aeruginosa, X-ray structure, microspectrophotometry.

Pseudomonas aeruginosa (Ps. aeruginosa) is a gram-negative bacterium pathogenic to both animals and plants (Zannoni (1989) Biochim. biophys Acta, 975, 299-316). It has been extensively studied and found to be resistant to most antibiotic families, due to the non-permeability of its outer membrane to antibiotics and its ability to metabolise them. In anaerobiosis, Ps. aeruginosa can grow using nitrate (NO3-) as the final electron acceptor in the respiratory chain and thus participates in the biogeochemical nitrogen cycle (Averill (1996) Chem. Rev., 96, 2951-2964)

Nitrite reductase (NiR) from Ps. aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing physiologically the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously calledThiosphaera pantotropha) (NiR-Pd) have been described (Fülop et al.(1995) Cell, 81, 369-377; Williams et al.(1997) Nature, 389, 406-412) and we recently reported on the structure of oxidized NiR-Pa at 2.15 A (Nurizzo et al., Structure (1997) 5, 1157-1171). Although the domains carrying the d1 heme are almost identical in both the oxidized and reduced structures of NiR-Pa and NiR-Pd, the c heme domains show different patterns of c heme coordination, depending on the species and the redox state. The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxyde ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd.

The structure of reduced NiR-Pa will be described in both the unbound (NiR-red) form and after binding with the physiological product, NO, at the d1 heme active site (NiR-NO). Although both structures are similar to that of oxidized NiR-Pa and to that of reduced NiR-Pd, significant differences were observed in two regions: firstly, a loop in the c heme domain (56-62) is displaced by 6 A; secondly, the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding, and thirdly the Tyr10 side-chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Even though the reaction pathway of the reduction are extremely different, it permit the accesibily of the catalytic site by the substrat.

The c heme is the the electron accepting pole and has been found in vitro to receive one electron from azurin or c551 with the same efficiency (Wharton et al.(1973), Biochim. Biophys. Acta 292, 611-620.; Silvestriniet al.(1982), Biochem. J., 203, 445-451) although the c551 is the physiological donor (Vijgenboom et al.(1997), Microbiol., 143, 2853-2863). Then the electron is transfered slowly to the d1 heme, the site of the reduction of nitrite. The NiR-red and NiR-NO models characterized by microspectrophotometry and X-ray crystallography show some structural differences due to the reduction of the both prosthetic groups. The reduction of the c heme was cryo-quenched at different stages, the degree of reduction being determined by microspectrophotometry. The frozen crystals were then exposed to X-rays to obtain structural information. In NiR-Pa, The conformational switches mentioned above in c heme domain seem to be only due to the reduction of the d1 heme since no conformational changes have been observed after reduction of the c heme only.

  1. Zannoni, D. (1989) The respiratory chains of pathogenic Pseudomonads. Biochim. Biophys. Acta 975, 299-316.
  2. Averill, B.A. (1996) Dissimilatory nitrite and nitric oxide reductases. Chem. Rev. 96, 2951-2964.
  3. Fülöp, V., Moir, J.W.B., Ferguson, S.J. & Hajdu, J. (1995) The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1. Cell, 81, 369-377.
  4. Williams, P.A., Fülop, V., Garman, E.F., Saunders, N.F.W., Ferguson, S.J. and Hajdu, J. (1997) Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme. Nature 389, 406-412.
  5. Nurizzo, D., Silvestrini, M.C., Mathieu, M., Cutruzzola, F., Bourgeois, D., Fülop, V., Hajdu, J. , Brunori, M., Tegoni, M. and Cambillau, C. (1997) N-terminal exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa . Structure 5, 1157-1171.
  6. Vijgenboom, E., Busch, J.E. and Canters, G.W. (1997) In vivo studies disprove an obligatory role of azurin in denitrification in Pseudomonas aeruginosa and show that azu expression is under control of RpoS and ANR. Microbiol. 143, 2853-2863.
  7. Wharton, D.C., Gudat, J.C. & Gibson, Q.H. (1973) Cytochrome oxidase from Pseudomonas aeruginosa - reaction with copper protein. Biochim. Biophys. Acta 292, 611-620.
  8. Silvestrini, M.C., Tordi, M.G., Colosimo, A., Antonini, E. & Brunori, M. (1982) The kinetics of electron transfer between Pseudomonas aeruginosa cytochrome c551 and its oxidase. Biochem. J. 203, 445-451.