The oxidation of hydroxylamine was studied by quantum chemical modeling. Hydroxylamine is the product of ammonia oxidation in ammonia monooxygenase. That mechanism has been studied recently by quantum chemical modeling as here. Only two enzymes can oxidize hydroxylamine, hydroxylamine oxidase and cytochrome-P460. Both employ the unusual P460-heme cofactor. In hydroxylamine oxidase, there is a covalently linked tyrosine, while in cytochrome-P460, there is a covalently linked lysine. The calculations give explanations for the experimental findings that NO is the final product in hydroxylamine oxidase, while N2O is the final product in cytochrome-P460. The effect of the covalent attachments has been investigated, and reasons for their presence have been given. The methodology used, which was proven to give very good agreement with experiments for several redox enzymes, again leads to excellent agreement with experimental findings.

Cytochrome c nitrite reductase (CcNiR) activates nitrite and produces ammonia. It is one of several enzymes that use a redox-active cofactor to perform its reaction. In this case, the cofactor has a heme with a lysine as the proximal ligand and a charged nearby arginine. The role of a tyrosine, which is also close, has been less clear. There are also four bis-histidine-ligated hemes involved in the electron transfers. CcNiR has been studied before, using essentially the same methods as here. However, the mechanism is very complicated, involving six reductions, and quite different results for the mechanism have been obtained here. For example, the tyrosine has here been found to be redox active in the final step when ammonia is produced. Also, the arginine has here been found to stay protonated throughout the mechanism, which is different from what was found in the previous study. The present results are in very good agreement with experimental findings and are, therefore, another case where the methodology has been shown to work very well. Previous examples include Photosystem II and Nitrogenase, normally considered to be the most important enzymes in nature for the development of life.

The mechanism for N-2 activation in the E-4 state of nitrogenase was investigated by model calculations. In the experimentally suggested mechanism, the E-4 state is obtained after four reductions to the ground state. In a recent theoretical study, results for a different mechanism have been found in excellent agreement with available Electron Paramagnetic Resonance (EPR) experiments for E-4. The two hydrides in E-4 leave as H-2 concertedly with the binding of N-2. The mechanism suggested differs from the experimentally suggested one by a requirement for four activation steps prior to catalysis. In the present study, the experimentally suggested mechanism is studied using the same methods as those used in the previous study on the theoretical mechanism. The computed results make it very unlikely that a structure obtained after four reductions from the ground state has two hydrides, and the experimentally suggested mechanism does therefore not agree with the EPR experiments for E-4. Another structure with only one hydride is here suggested to be the one that has been observed to bind N-2 after only four reductions of the ground state.

Nitrogenase converts nitrogen in the air to ammonia. It is often regarded as the second most important enzyme in nature after photosystem II. The mechanism for how nitrogenase is able to perform the difficult task of cleaving the strong bond in N2 is debated. It is known that for every electron that is donated to N2, two ATP are hydrolyzed. In the experimentally suggested mechanism, the activation occurs after four reductions of the ground state, but there is no suggestion for how the enzyme uses the hydrolysis energy to perform catalysis. In the theoretical mechanism, it is suggested that hydrolysis is used to reduce the electron donor. In previous papers, the steps leading to the activation of N2 in the so-called E4 state has been investigated, using both the experimental and theoretical mechanism, showing that only the theoretical one leads to agreement with EPR observations for E4. In the present paper, the four steps following E4, leading to the release of two ammonia molecules, are described using the same methodology as used in the previous studies.

Particulate MMO (pMMO) catalyzes the oxidation of methane to methanol and also ammonia to hydroxylamine. Experimental characterization of the active site has been very difficult partly because the enzyme is membrane-bound. However, recently, there has been major progress mainly through the use of cryogenic electron microscopy (cryoEM). Electron paramagnetic resonance (EPR) and X-ray spectroscopy have also been employed. Surprisingly, the active site has only one copper. There are two histidine ligands and one asparagine ligand, and the active site is surrounded by phenyl alanines but no charged amino acids in the close surrounding. The present study is the first quantum chemical study using a model of that active site (Cu-D). Low barrier mechanisms have been found, where an important part is that there are two initial proton-coupled electron transfer steps to a bound O-2 ligand before the substrate enters. Surprisingly, this leads to large radical character for the oxygens even though they are protonated. That result is very important for the ability to accept a proton from the substrates. Methods have been used which have been thoroughly tested for redox enzyme mechanisms.

Nitrogenases are the only enzymes that activate the strong triple bond in N2. The mechanism for the activation has been very difficult to determine in spite of decades of work. In previous modeling studies it has been suggested that the mechanism for nitrogen activation starts out by four pre-activation steps (A0–A4) before catalysis. That suggestion led to excellent agreement with experimental Elecrtron Paramagnetic Resonance (EPR) observations in the step where N2 becomes protonated (E4). An important part of the pre-activation is that a sulfide is released. In the present paper, the details of the pre-activation are modeled, including the release of the sulfide. Several possible transition states for the release have been obtained. An A4(E0) state is reached which is very similar to the E4 state. For completeness, the steps going back from A4(E0) to A0 after catalysis are also modeled, including the insertion of a sulfide.

The main class of nitrogenases has a molybdenum in its cofactor. A mechanism for Mo-nitrogenase has recently been described. In the present study, another class of nitrogenases has been studied, the one with a vanadium instead of a molybdenum in its cofactor. It is generally believed that these classes use the same general mechanism to activate nitrogen. The same methodology has been used here as the one used for Mo-nitrogenase. N₂ activation is known to occur after four reductions in the catalytic cycle, in the E₄ state. The main features of the mechanism for Mo-nitrogenase found in the previous study are an activation process in four steps prior to catalysis, the release of a sulfide during the activation steps and the formation of H₂ from two hydrides in E₄, just before N₂ is activated. The same features have been found here for V-nitrogenase. A difference is that five steps are needed in the activation process, which explains why the ground state of V-nitrogenase is a triplet (even number) and the one for Mo-nitrogenase is a quartet (odd number). The reason an additional step is needed for V-nitrogenase is that V³⁺ can be reduced to V²⁺, in contrast to the case for Mo³⁺ in Mo-nitrogenase. The fact that V³⁺ is Jahn–Teller active has important consequences. N₂H₂ is formed in E₄ with reasonably small barriers.

Model calculations have been performed for the singly reduced ground state of Mo-nitrogenase, usually termed E₁. Contradictory conclusions have been reached in two recent experimental studies. In a study based on EPR, it was concluded that there is a bridging hydride in E₁, while in an X-ray study it was concluded that there is no hydride in E₁. Therefore, the EPR study implies that there is an oxidation of the cofactor going from E₀ to E₁, the X-ray study implies a reduction. DFT methods have here been used, which have previously been benchmarked on a set of redox enzymes that led to the conclusion that the accuracy is about 3 kcal mol⁻¹ in all cases, even for redox transitions. The methodology should therefore be adequate for resolving the question of the hydride presence in E₁. As a comparison, calculations are performed on both Mo- and V-nitrogenase with the same conclusion. The conclusion from the calculations has far reaching consequences for the mechanism of nitrogenase.

A computational methodology is briefly described, which appears to be able to accurately describe the mechanisms of redox active enzymes. The method is built on hybrid density functional theory where the inclusion of a fraction of exact exchange is critical. Two examples of where the methodology has been applied are described. The first example is the mechanism for water oxidation in photosystem II, and the second one is the mechanism for N2 activation by nitrogenase. The mechanism for PSII has obtained very strong support from subsequent experiments. For nitrogenase, the calculations suggest that there should be an activation process prior to catalysis, which is still strongly debated. 

When moving protons in enzymes, water molecules are often used as intermediates. The water molecules used are not necessarily seen in the crystal structures if they move around at high rates. In a different situation, for metal containing cofactors in enzymes, it is sometimes necessary to move protons on the cofactor from the position they enter the cofactor to another position where the energy is lower. That is, for example, the situation in nitrogenase. In recent studies on that enzyme, prohibitively high barriers were sometimes found for transferring protons, and that was used as a strong argument against mechanisms where a sulfide is lost in the mechanism. A high barrier could be due to nonoptimal distances and angles at the transition state. In the present study, possibilities are investigated to use water molecules to reduce these barriers. The study is very general and could have been done for many other enzymes. The effect of water was found to be very large in the case of nitrogenase with a lowering of one barrier from 15.6 kcal/mol down to essentially zero. It is concluded that the effect of water molecules must be taken into account for meaningful results.