This Is AuburnElectronic Theses and Dissertations

Methyl-coenzyme M reductase: Expressing active recombinant enzyme in M. maripaludis, and investigating the activation process, and the mechanism of inhibition of 3-nitrooxypropanol and Studying the effect of Ionic liquids on the mononuclear rearrangement of heterocycles using QM/MM method




Ghebreab, Robel

Type of Degree

PhD Dissertation


Chemistry and Biochemistry


The study of the biosynthesis of methane is appealing from both its potential role in biofuel production and environmental perspectives. Methane is the main component of natural gas, and it is also a potent greenhouse gas as it is the second most abundant greenhouse gas after CO2. Methane is produced through geothermal processes and through the action of microorganisms known as methanogenic Archaea. Methanotrophic Archaea on the other hand are involved in the biological oxidation of methane. Together with aerobic methanotrophs, methanotrophic Archaea play an essential role in preventing the release of methane to the atmosphere which otherwise could have a serious contribution to global warming and climate change.1–3 Methyl-coenzyme M reductase (MCR) is the key enzyme in both the biological formation and the anaerobic oxidation of methane. MCR catalyzes the terminal step in methane formation and first step in the oxidation of methane. MCR catalyzes the reversible reaction of methyl-coenzyme M and coenzyme B to produce methane and the corresponding heterodisulfide. The catalytic activity of MCR depends on its unique nickel-containing tetrapyrrole core, coenzyme F430, which can exist in three different oxidation states. MCR exhibits nickel-based EPR signals when the nickel center is either in the oxidation state +1 or +3. This enzyme is active in the oxidation state +1 and exhibits an axial EPR signal, called MCR-red1. In the presence of coenzyme M and coenzyme B, the enzyme exhibits a rhombic signal, MCR-red2. Two other paramagnetic states of the enzyme can be detected, MCR-ox1 and MCR-ox2. The Nickel center has an oxidation state of +3 in these states. The ox states are inactive, but can be converted in vitro to the active state of the enzyme, MCR-red1, by treating it with Ti(III) citrate. Near its active site, MCR has five post-translational modifications (PTMs). Four of these five modifications are methylations, including 2-(S)-methylglutamine, 5-(S)-methylarginine, 3-methylhistidine, S-methylcysteine, and the fifth modified amino acid is a thioglycine in which the backbone carbonyl oxygen is replaced with sulfur. The exact roles of these PTMs are not known, but the amino acids are believed to have some role in the catalytic mechanism of the enzyme. Moreover, a large multi protein complex is required for the activation of MCR in a process that involves both electron bifurcation and ATP hydrolysis. The research reported here was aimed at getting a more thorough understanding of the expression, activation, and inhibition of MCR. A general introduction is presented in chapter one. The second chapter describes the expression and characterization of recombinant MCRs in Methanococcus maripaludis. The main reason to use M. maripaludis as a host for expressing MCR was that MCR requires several genes to correctly express including, but not limited to, genes needed for PTMs, genes required for activating MCR, and genes required for the biosynthesis of F430. Most of them are not yet known. Thus, there could be a higher chance of success when expressing MCR in Archaea which contain all the machinery for getting mature and active MCR. MCR from Methanothermococcus okinawensis and from M. maripaludis were cloned and expressed in M. maripaludis. Surprisingly, the recombinant MCR from both organisms were purified into two distinct fractions. One set of fractions was without F430 and yet contains McrD. The function of McrD is not known, thus we proposed it may be involved in the insertion of F430 to apo-MCR. The second fraction contained the expected MCR with F430, but no McrD was detected in this fraction. This raised several questions and the chapter presents some of them in detail. The third chapter “Activation system of MCR” describes the investigation of the MCR activation system from M. marburgensis. In vivo, MCR is activated by a large complex referred to as A3a. This chapter extends the investigation of this activating complex. In the fourth chapter the inhibitory mechanism of 3-NOP on MCR is investigated. Large quantities of methane are produced and escaped to the atmosphere from dairy cows and other ruminal livestock. This not only is a burden to the atmosphere, but also a significant amount of nutrition is lost during methane biosynthesis. This loss could be reduced by inhibition of MCR. The chapter discusses the inhibitory mechanism of 3-NOP, a small molecule of MCR inhibitor developed by DSM. Chapter five briefly introduces the computational chemistry. The following chapter then presents computationally obtained data on the effect of ionic liquids on the mononuclear rearrangement of heterocycles (MRH). Mixed quantum mechanics/molecular mechanics (QM/MM) combined with free-energy perturbation theory (FEP) and Monte Carlo (MC) sampling was used to investigate the nature of the rate enhancement in ionic liquids in this type of reaction. The geometry and activation energy barrier of rearrangement of the Z-phenylhydrazone of 3-benzoyl-5-phenyl-1,2,4-oxadiazole into 4-benzoyla-mino-2,5-diphenyl-1,2,3-triazole in the 1-butyl- 3-methylimidazolium tetrafluoroborate [BMIM] based ionic liquid as well as in conventional organic solvents, methanol, and acetonitrile were computed for both uncatalyzed and general base catalyzed reactions. Detailed analysis is also presented to explain the origin of rate enhancement observed in [BF4] ionic liquid. A significant difference in the activation barriers were observed, not only between conventional solvent and ionic liquids, but also among the two ionic liquids used for this study. The rate enhancement observed in [BMIM][BF4] could not be attributed to the physical properties of the solvents used, (i.e., to the polarities or viscosity of the solvent) as the two ionic liquid have comparable properties. The computational results and analysis of structure and energetics of the reaction suggest that the difference in rate enhancement lies in the geometric arrangement of the ionic liquids at the transition state. A favorable π+-π interaction between the imidazolium ring of the BMIM cation and the bicyclic quasi-aromatic 10π oxadiazole/triazole transition state region of the substrate was observed. This imposed a pre-ordered geometric arrangement that stabilize the transition structure, lowering the activation barrier, and thereby enhancing the rate of the rearrangement reaction. A coplanar orientation of the phenyl rings that maximized the electronic effects exerted on the reaction route together with site-specific electrostatic stabilization between the ions and the MRH substrate in [BMIM][BF4] as compared to [BMIM][PF6] could explain the rate enhancement observed in [BMIM][BF4].