N-TERMINAL ARM EXCHANGE IS OBSERVED IN THE 2.15-ANGSTROM CRYSTAL-STRUCTURE OF OXIDIZED NITRITE REDUCTASE FROM PSEUDOMONAS-AERUGINOSA

Background: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which con tains one c and one d(1) heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy , can be induced by the addition of nitrate to the bacterial growth me dium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c(551) and azurin are efficient elect ron donors in this reaction. NIR is a key denitrification enzyme, whic h controls the rate of the production of toxic nitric oxide (NO) and u ltimately regulates the release of NO into the atmosphere. Results: Th e structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa w as solved at 2.15 Angstrom resolution, using molecular replacement wit h the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d(1)-heme domains are almost identica l in both enzyme structures, the c domain of NiR-Pa is more like the c lassical class I cytochrome-c fold because it has His51 and Met88 as h eme ligands, instead of His17 and His69 present in NiR-Tp. In addition , the methionine-bearing loop, which was displaced by His17 of the NiR -Tp N-terminal segment, is back to normal in our structure. The N-term inal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence ident ity. In Nir-Pa, this N-terminal segment of one monomer crosses the dim er interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion - the sixth ligand of t he d(1)-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is d irectly bound to the Fe. Conclusions: Two ligands of hemes c and d(1) differ between the two known NIR structures, which accounts for the fa ct that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously o bserved in other systems and may explain the observed cooperativity be tween monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas st utzeri, which has an N terminus very different from that of the two ab ove mentioned NiRs.