Dynamic Hydrogen-Bonding Network in the Distal Pocket of the Nitrosyl Complex of Pseudomonas aeruginosa cd₁ Nitrite Reductase

Abstract

cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d(1)-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 d(1)-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 d(1)-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 d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) 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 (Tyr(10), His(327), and His(369)), 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 d(1)-heme complex of cd(1) NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) 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, His(369) is closer to the NO, whereas mutation of both distal histidines displaces Tyr(10), removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.

Mononuclear–Dinuclear Equilibrium of Grafted Copper Complexes Confined in the Nanochannels of MCM‐41 Silica

“…Recently, a proton delivery channel was identified in the resting state of nitrite reductase (NiR) enzyme: the His-255 residue near the type II (T 2 )Cu active site is protonated and hydrogen-bonded to a negatively charged Asp-98 residue through a water molecule. [74][75][76] Therefore, in the present case, proton transfer can occur along the ÀOH···ClO 4 À ··· + H 3 NÀ hydrogen-bonding network in the presence of water, which explains the presence of a mononuclear-dinuclear equilibrium of complexes with perchlorate ions. Based on the characterization by a combination of EPR, UV/Vis, FTIR, and TGA, we conclude that perchlorate serves as a hydrogen-bond network maker, not as a ligand for copper, whereas chloride serves as a good ligand for copper.…”

Regulating the nitrite reductase activity of myoglobin by redesigning the heme active center

“…7c), suggesting a conformational change for the axial NO by eliminating its interaction with the distal His64. Mutagenesis studies of native NIR also confirmed that the conserved distal histidines, especially for His327, are involved in formation of the nitrosyl-heme d 1 complex and determine its stability [56].…”

“…Although a true channel may not be generated in the native enzyme, conformational changes occurred for the distal Tyr10 upon nitrite reduction that rotates away from the position adopted in the oxidized form [58]. Moreover, it was shown that the hydrogen-bonding network is dynamic in the heme d 1 pocket, which facilitates ligand binding and product release [56]. For Mb system, the removal of distal His64 also altered the distal hydrogenbonding network ( Fig.…”

The role of porphyrin peripheral substituents in determining the reactivities of ferrous nitrosyl species

“…9 Alternatively, Cd 1 NiR forms {FeNO} 7 through an electron transfer (ET) from cytochrome c, and releases NO with the concomitant binding of nitrite to the ferrous heme-d 1 and the cycle continues. [12][13][14] The different reactivity of {FeNO} 7 species compels investigating the difference in the active sites that controls the competition between the PCET process and NO release. A {FeNO} 7 adduct generally possesses a very strong Fe-NO bond with a K d $ 10 À9 and this displacement of NO by nitrite is rather unexpected.…”