Quantum Mechanical Interpretation of Nitrite Reduction by Cytochrome cd₁ Nitrite Reductase from Paracoccus pantotrophus

Abstract

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fülöp, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fülöp, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fülöp, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.

The nitrite reductase from Pseudomonas aeruginosa : Essential role of two active-site histidines in the catalytic and structural properties

“…We thus propose that substitution of either one of the two invariant active-site His with Ala and reduction of the positive potential in the pocket is associated with loss of the hydroxyl, which was found to be coordinated with the ferric d 1 -heme iron in the wt enzyme (4) and should assist NO dissociation. This hypothesis is in agreement with quantum mechanical calculations (29), indicating that interaction with the active-site His can stabilize and orient nucleophilic ligands of the d 1 -heme, the presence of which imposes an unfavorable orientation on NO and thereby facilitates its release. It is worth noting that it has been recently shown that a buried carboxylate in the active site of nitrophorins (a class of heme proteins with vasodilatory activity associated with NO release) promotes dissociation of NO from the ferric heme iron (30).…”

The Structure of an Alternative Form ofParacoccus pantotrophus Cytochromecd 1 Nitrite Reductase

“…the movement of the tethered shuttle protein) either in catalysis (2,4,27) or in the control of catalysis (4,5,11,27). Although the presence of alternative conformers of NiR has long been a subject of speculation, the dramatic alteration in relative domain arrangement compared with the previously known structures was unexpected.…”

Structure and Kinetic Properties of Paracoccus pantotrophus Cytochrome cd1 Nitrite Reductase with the d1 Heme Active Site Ligand Tyrosine 25 Replaced by Serine

“…However, it is still possible, for example, that approach of tyrosine 25 toward the two active-site histidine residues (positions 345 and 388) plays a role in driving dissociation of nitric oxide (17) and that this role can be replicated by the hydroxy group of serine 25. In other words, as discussed elsewhere (17) (17).…”

“…17). Structures of cytochrome cd 1 from crystals that had first been reduced and then treated with nitrite showed tyrosine 25 apparently poised ready to displace a bound nitric oxide (9); the significance of this residue, but not necessarily its religation to the d 1 heme iron, for this displacement has been indicated by calculations (17). The driving force for tyrosine 25 to ligate to the oxidized iron of the d 1 heme is certainly large.…”