Computational Studies of Metalloenzymes

Enzymes are involved in most reactions in nature. They are important both for the understanding of biological life and for reactions of industrial interest, e.g. in the production of artificial fertilizers, the production of biomass or biofuels. Enzymes with one or several metal ions are called metalloenzymes. In this thesis we study three different metalloenzymes, nitrogenase, lytic polysaccharide monooxygenase (LPMO) and particulate methane monooxygenase (pMMO) with computer simulations. We use mainly combined quantum mechanical and molecular mechanical (QM/MM) calculations with the density functional theory (DFT) method.

Nitrogenase is the only enzyme that can break the triple bond in nitrogen molecules, making nitrogen available for plant metabolism. Previous studies have shown that different DFT methods give widely different results for the relative energies of different structures of putative intermediates in the reaction mechanism of nitrogenase. Therefore, we have tried to calibrate DFT calculations in two different ways. First, we use experimental data of structures and reactions related to nitrogenase to see what DFT functionals give the most accurate results.
Our results indicate that BLYP, B97D and MN15 give the best results.
Second, we have developed a small and simple model system of the active site of nitrogenase, [Fe(SH)4H]−.

This model still shows a large variation in DFT estimate of the energy difference between structures protonated on Fe or on S, 25–163 kJ/mol. We then use a series of advanced and accurate QM methods, including coupled-cluster, selected configuration-interaction and multicon gurational perturbation theory methods to calibrate the DFT methods. With this model, M06 and B3LYP give the most accurate results.

The second studied enzyme is LPMO, which is a copper dependent enzyme used for the degradation of polysaccharides, such as cellulose and chitin. The mechanism of this enzyme is quite well known but it has an unusual methyl modi cation of one of the ligands. Our calculations suggest that this group may protect the enzyme from self-oxidation.

The third studied enzyme is pMMO. This enzyme can hydroxylate methane. This enzyme is hard to study experimentally and the nature and location of the active site is still controversial. We have studied the reactivity of three putative active sites, involving mononuclear copper sites, and have shown that they
can support similar reactions. The CuC site gives the most favourable energetics.

Enzymes are important for many reactions in nature. They are proteins that increase the speed of chemical reactions without reacting themselves. Thus, they catalyze the reactions. In the reactions, the enzymes act on other molecules called substrates. The created molecules are called products. Almost all processes in a living cell are catalyzed by enzymes ensuring that they are fast enough. In the human body alone, there are more than 700 different enzymes. Enzymes are important also for industrial applications e. g. the production of bio-fuels and artificial fertilizers. Hence, it is important to understand how enzymes work.

Both experiment and computer simulations are used to study enzymes. The comp uter power has increased much during the last decades and therefore computer simulations have become increasingly important. The fundamental theories behind the simulations are older.

For objects we meet in daily life we know which physical rules applies. They are called classical mechanics. For much smaller particles, partly others, sometimes surprising, rules are relevant. This is called quantum mechanics.

Quantum mechanics is the foundation of what we today call quantum chemistry. The theory was developed by researches around 100 years ago. According to quantum mechanics, electrons are rotating around atomic nuclei and only certain energy levels are allowed, the energy is quantized. To find the energy of an electron we have to solve the so-called Schrödinger equation. It is what the mathematicians call a partial differential equation, i. e. an equation containing derivatives and the solution is a function of several variables. The variables are the time and the coordinates of all particles. The solutions are called wave functions and indicate the probability that a particle is at a certain position.

Unfortunately, the Schrödinger equation can be solved analytically only for a few simple cases. Therefore simplifcations are needed to be able to solve the Schrödinger equation with a computer. This is called numerical solutions.
In this thesis, we use mainly two numerical methods, molecular mechanics and density functional theory (DFT). DFT states that energy of the ground state can be found as a functional of the electron density

We have used these methods to study three different enzymes. Nitrogenase is the only enzyme that can cleave the strong triple bond in molecular nitrogen to form two molecules of ammonia and make atmospheric nitrogen available to plant life. It contains in its active site a complicated MoFe7S9C cluster, called the FeMo cluster. Previous DFT investigations have shown that the results depend strongly on which DFT method is used, so that it is not known which method can be trusted. Therefore, we have used two different approaches to calibrate the DFT calculations. In the first, we use experimental data of structures and reactions related to nitrogenase. In the second, we instead use more advanced quantum mechanical methods on a minimal model of the FeMo cluster.

Lytic polysaccharide monooxygenase is an enzyme that degrades cellulose by an oxidative reaction. The active site contains a copper ion bound two histidine ligands. One of them often has an unusual methylation, but the reason for this modication has been unknown. Our calculations show that this group most likely protects the enzyme against self-oxidation.

Particulate methane monooxygenase (pMMO) is one of two enzymes that can hydroxylate methane, forming methanol, which can be used as a more convenient fuel than natural gas. The enzyme is membrane-bound and therefore hard to study experimentally. Therefore, the nature and position of the active site is still not known. We have studied the reactivity of three putative active sites in pMMO, all mononuclear copper sites. We show that all three sites can perform similar chemical reactions, but only one of them, the CuC site, has favourable energetics.