CONFORMATIONAL-CHANGES OCCURRING UPON REDUCTION AND NO BINDING IN NITRITE REDUCTASE FROM PSEUDOMONAS-AERUGINOSA

Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR- Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each mo nomer carries one c and one d(1) heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphae ra pantotropha) (NiR-Pd) have been described [Fulop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412] , and we recently reported on the structure of oxidized NiR-Pa at 2.15 Angstrom [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Althoug h the domains carrying the d(1) heme are almost identical in both NiR- Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d(1) 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 hydroxide ion. Furthermore, upo n reduction, the axial ligand of the c heme of NiR-Pd changes from His 17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-termina l stretch of residues (1-29) of one monomer interacts with the other m onomer (domain swapping), which does not occur in NiR-Pd. Here the str ucture of reduced NiR-Pa is described both in the unbound form and wit h the physiological product, NO, bound at the d(1) heme active site. A lthough both structures are similar to that of reduced NiR-Pd, signifi cant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 Angstrom away and (ii) the hydroxide ion, which is the sixth coordina tion ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxi dized form. The conformational changes observed in NiR-Pa as the resul t of reduction are less extensive than those occurring in NiR-Pd. Star ting with oxidized structures that differ in many respects, the two en zymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involve d in catalysis are considerably diverse.