Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

Abstract

Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.

Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase: Insights from Density Functional Theory Studies

“…To construct the scheme, an adiabatic electron affinity of 94 kcal/mol and the proton absolute hydration enthalpy from cluster-ion solvation (−275 kcal/mol) 86 were used as reference values. 111 The scheme contains protonation energies, electron affinities, and bond formation energies, as well as calculated pK a values and redox potentials. These values were estimated from the free energy differences ΔG using equations presented in section 1.4.1.…”

“…Therefore, it was postulated that the calcium site may act as an acid/base catalytic subunit. 111 The protons from the dissociated coordinated water molecules can be directly transferred to the active site because the substrate molecule is only few angstroms away from the calcium coordination sphere. There are free energy changes (ΔG in kcal/mol) in the square brackets and relative energies with respect to the initial (NO) complex in the curved brackets.…”

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

“…With two protons from the distal residues, a molecule of water is released, forming a {FeNO} 6 intermediate (Enemark-Feltham notation). 5,9,10 The CcNiR avoids the formation of the dead-end intermediate, {FeNO} 7 through two consecutive proton-coupled electron transfer (PCET) to the {FeNO} 6 species, generating an {FeHNO} 8 intermediate, 11 which, on further reduction, leads to the generation of NH 4 + . 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.…”

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochromecnitrite reductase

“…Based on the fact that the O atom of NO is bound to His361 and Tyr303, it can be assumed that the first nitrite O atom to be cleaved is that bound to Arg131, which corresponds to a variant of the catalysis discussed by Bykov & Neese (2012). On the other hand, elimination of the nitrite O atom forming hydrogen bonds to His361 and Tyr303 seems to be more likely because His and Tyr residues are much more effective proton donors than an Arg residue.…”