Domain swing upon his to ala mutation in nitrite reductase of Pseudomonas aeruginosa

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a solub le enzyme catalysing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme dom ain and a d(1)-heme domain. The structures of the enzyme in both the oxidis ed and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-direct ed mutants has shown that the substitution of either one of these two His w ith Ala dramatically reduces the physiologically relevant reactivity toward s nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H36 9A in complex with NO have been solved by multiple wavelength anomalous dis persion (MAD), using the iron anomalous signal, and molecular replacement t echniques. In both refined crystal structures the c-heme domain, whilst pre serving its classical c-type cytochrome fold, has undergone a 60 degrees ri gid-body rotation around an axis parallel with the pseudo 8-fold axis of th e beta -propeller, and passing through residue Gln115. Even though the dist ance between the Fe ions of the c and d(1)-heme remains 21 Angstrom, the ed ge-to-edge distance between the two hemes has increased by 5 Angstrom. Furt hermore the distal side of the d(1)-heme pocket appears to have undergone s tructural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly diff erent from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the dist inction between nitrite and oxidase reduction mechanisms. Moreover they dem onstrate that the two histidine residues play a crucial role in the physiol ogical activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa. (C) 2001 Academic Pre ss.