要旨
cd1 nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d1-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d1-heme to avoid enzyme inhibition by the formation of ferrous−nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d1-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761−14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d1-heme NO complex from Pseudomonas aeruginosa cd1 NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the “distal” side of the heme (Tyr10, His327, and His369), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron−nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d1-heme complex of cd1 NIR forms H-bonds with Tyr10 and His369, whereas the second conserved histidine, His327, appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr10 is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His369 is closer to the NO, whereas mutation of both distal histidines displaces Tyr10, removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.
