Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

Abstract

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b(3)-Fe(B) active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe(B) consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu(B) in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b(3) and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe(B) (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe(B) the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b(3) taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe(B) will form a mu-oxo bridge to heme b(3) in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable mu-oxo bridge between heme b(3) and Fe(B) is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe(B) in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…In synthetic models of NOR, it has also been found that the presence of a glutamic acid mimic significantly increases the stability of iron binding to the Fe B site (40). Furthermore, a theoretical study by Blomberg et al (58) showed that a model with an Fe B coordinated by three histidines, one glutamate, and one water molecule provides an energetically feasible reaction mechanism of NO reduction. However, the structural model of NOR constructed recently by Reimann et al (22) shows that the closest conserved Glu (E267) still has its carboxylate O atom 7 Å away from Fe B , which suggests that Glu may not bind to Fe B in native NOR.…”

Characterization of the nitric oxide reductase from Thermus thermophilus

“…In addition, computational (42,43) as well as modeling studies (44) on NOR activity have emphasized the importance of the negative charge provided by E211 to NOR activity. One explanation for the relatively high activity of the E211A mutant is that the carboxylate group, at least in the TtcNOR, can be replaced by a hydroxyl (or water) ligand.…”

The bacterial respiratory nitric oxide reductase

“…It is proposed that, after the second NO molecule binds trans to the first, the reaction proceeds via a hyponitrite transition state [39,43] (Figure 2B). The participation of this hyponitrite species is also predicted from a computational study of the reaction mechanism based on the homology model of the P. stutzeri NorB which was adjusted to force Fe B to have octahedral co-ordination with the conserved Glu 211 (Glu 198 in P. denitrificans) to serve as an additional ligand [48].…”