In Silico Insight into the Reductive Nitrosylation of Ferric Hemeproteins

Abstract

A combination of in silico methods was used to extend the experimental description of the reductive nitrosylation mechanism in ferric hemeproteins with the molecular details of the role of surrounding amino acids. The computational strategy consisted in the estimation of potential energy profiles for the transition process associated with the interactions of the coordinated N(NO) moiety with O(H 2 O) or O(OH − ) as nucleophiles, and with distal amino acids as proton acceptors or affecting the stability of transition states. We inspected the reductive nitrosylation in three representative hemeproteins -sperm whale metmyoglobin, α subunit of human methemoglobin and nitrophorin 4 of Rhodnius prolixus. For each case, classical molecular dynamics simulations were performed in order to obtain relevant reactive conformations, and a potential energy profile for the reactive step was obtained using adiabatic mapping or nudged elastic band approaches at the QM/MM level. Specifically, we report the role of a charged Arg45 of myoglobin in destabilizing the transition state when H 2 O acts as nucleophile, differently to the neutral Pro43 of the hemoglobin subunit. The case of the nitrophorin is unique in that the access of the required water molecules is scarce, thus, preventing the reaction.

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…Although Mb and Hb contain a distal His residue, which acts as a gate for solvent access, the autoreduction reaction occurs under different conditions in these proteins . The computational work shows that the transition state for water acting as a nucleophile and attacking the NO + ligand in the Mb­(III)–NO adduct has an energy of 15 kcal/mol relative to the reactants, whereas, in contrast, the reaction with hydroxide is barrierless . This evidence supports previous experimental findings that autoreduction occurs at basic pH in Mb.…”

“…The presence of this residue destabilizes a hydrogen bond between water and the distal His in the transition state, resulting in an energy barrier of 15 kcal/mol . In contrast, the distal pocket of Hb contains a neutral proline, which does not affect the hydrogen bond between water and the distal His (at neutral pH), leading to a much lower energy barrier of only 5 kcal/mol for water attack on the Fe­(II)–NO + complex . Interestingly, these computational results are also in agreement with the lack of autoreduction activity in rNP at neutral pH.…”

“…In the next step, HNO 2 dissociates from the ferrous heme, followed by rapid binding of NO to form a ls-{FeNO} 7 complex (see Scheme ). , …”

“…The higher energy barrier at neutral pH is due to a nearby positively charged Arg45 residue. The presence of this residue destabilizes a hydrogen bond between water and the distal His in the transition state, resulting in an energy barrier of 15 kcal/mol . In contrast, the distal pocket of Hb contains a neutral proline, which does not affect the hydrogen bond between water and the distal His (at neutral pH), leading to a much lower energy barrier of only 5 kcal/mol for water attack on the Fe­(II)–NO + complex .…”

A Copper(II)‐Nitrite Complex Hydrogen‐Bonded to a Protonated Amine in the Second‐Coordination‐Sphere

“… [8,9] Moreover, the heme‐ferrous site of deoxy‐hemoglobin (deoxy‐Hb) reduces NO 2 − and affords nitrosyl‐Hb through the involvement of an Fe‐bound nitrous acid species [4,10] . The intermediacy of a heme‐Fe‐(HONO) species has also been proposed in the reductive nitrosylation of methemoglobin [11,12] . The active sites of nitrite reductases (NiRs) and hemoglobin (Hb) possess proton‐responsive amino acid residues such as histidine (His) and aspartate (Asp) in the second‐coordination‐sphere (Figure 1), [4,9] thereby assisting proton transfer to the metal‐bound nitrite anion as well as stabilizing the metal‐nitrous acid intermediate.…”