Research Interests
 

Biological Activation of Small Molecules

        One of the most tantalizing challenges at the interface of biology and chemistry is the understanding of complex biochemical processes in which inert molecules1,2, such as H2, N2 and CO2, are transformed into more reactive forms, such as protons, electrons, ammonia, and methane. These transformations occur at ambient temperature and pressure; therefore their technological implementation would be valuable. Beyond the low energy need of these processes, these 'green chemical' transformations - developed by learning from Nature - would be environmentally sound, hazardous waste-free technologies.

          The objective of the proposed work is to investigate three coupled biochemical processes, such as nitrogen fixation, hydrogen evolution and up take, and methanogenesis (Figure 1), present in most anaerobic bacteria.
 
Figure 1		         The nitrogen-3,4 and hydrogen-cycles5 (N- and H-cycles) are structurally well characterized and currently the focus of intensive spectroscopic investigation. Computational modeling studies have been carried out6-12 to simulate selected intermediates of the reaction cycles. Due to the complexity of the active-site modeling, most of the simulations are restricted to simplified, hypothetical model complexes in the gas phase. Despite more than three decades of experimental and about a decade of computational investigations, the molecular mechanism of both the N- and the H-cycles are yet to be understood. The full carbon-cycle13,14 (C-cycle) is less defined at the molecular level, and most of the work has focused on the final step15 of methanogenesis, which is the methane evolution from cofactor F430. An understanding of the complete carbon dioxide conversion to methane would allow for the definition of alternative activation pathways and novel biomimetic model complexes with industrial potential.
          N-cycle: The active-site of the nitrogenase metalloenzyme is the so-called iron-molybdenum-cofactor (FeMo-co), where the N2 binding and reduction occur (Figure 2). It was structurally characterized16-22 about a decade ago, which intensified the research of the molecular mechanism. Alternative nitrogenases23 are also known in which the molybdenum ion is replaced by vanadium or iron ions with lower nitrogen fixation activity24. The protein bound FeMo-co has a rich activation chemistry25, since it is capable of reducing the N-N, C-N, C-C triple and double bonds. Furthermore, non-competitive inhibition studies26 indicate multiple substrate binding sites.
  The extracted FeMo-co from the protein matrix can be obtained in a reversible process27 in N-methyl-formamide solvent. The structure of the extracted cofactor28,29 and the reversible extraction process are not yet well understood. Moreover, side-directed point-mutations30 revealed an intricate role of the protein environment. A slight change in the amino acid residues around the cofactor can lead to the loss of the nitrogen fixation activity, while alternative substrates can bind and be converted to their reduced form by the mutant enzymes. In addition, chemical modifications revealed the critical role of the homocitrate ligand31, which is bound to the molybdenum site of the cluster. If it is replaced by a citrate ligand, the nitrogen fixation activity is reduced by nearly two orders of magnitude. The molecular bases of these environmental effects are yet to be defined.
Figure 2
          Proposed research: The starting point of the proposed research is a combined computational and XAS study of well defined model compounds32-34, which contain the essential [Mo/V/Fe-Fe3S4] heterometal cube with similar ligands to that of the FeMo-co. The Mo-clusters are able to reduce compounds with single and double N,N bonds, such as hydrazine and diazyne, respectively, however they cannot reduce dinitrogen to ammonia. The DFT potential energy surface description of these heterometal cubane clusters could reveal the reason for the reactivity difference of protein bound FeMo-co and the biomimetic model compounds. Reactants, products and isolatable intermediates will be synthesized and characterized by metal (Fe, Mo) and ligand K-edge (S,Cl), as well as, metal (Mo) L-edge X-ray absorption spectroscopies. In addition, using molecular mechanical and semi-empricial computational methods, the oligomeric structure of the extracted FeMo-co will be simulated based on the X-ray structure of Fe-S clusters and EXAFS geometry parameters, which have already been determined. Employing high accuracy protein forcefields, such as AMBER or CHARMM, the structural changes introduced by mutations and the role of the protein environment in nitrogenase function will be calculated using the structures of apo- and holoproteins. The structural effects and potential interactions with the FeMo-co will be experimentally verified by EXAFS studies as mutant enzyme samples become available from collaborators.
 
Figure 3		         H-cycle: The hydrogenases are the essential enzymes in the H-cycle and responsible for H2 reduction and oxidation reactions5. Three major types of enzymes have been characterized so far, which are the nickel-iron35,36, the iron-only metalloproteins37-39 and a group of enzymes40 that do not contain a bound transition metal. The proposed research focuses on the iron-only hydrogenase, first characterized structurally from Clostridium pasteurianum41,42. In this organism, the H-cycle is coupled to the nitrogenase function, since the evolved H2; as a sideproduct to NH3 is reduced to protons and electrons by hydrogenases that are utilized in the nitrogen reduction. The active-site structure (Figure 3) has revealed a biochemically unusual coordination of Fe ions with cyanide and carbonyl ligands. The [4Fe-4S] cluster, which is assumed to be the electron buffer for the redox chemistry, can donate or accept electrons formed in H2 evolution or uptake, respectively. The H-cluster, which is linked through a cysteine residue to the tetranuclear cluster, is the site where the heterolytic cleavage/formation of the H,H bond occurs. The involvement of the terminal iron in the reaction mechanism is supported by experiment43-45 and theoretical simulation46-50; however, the roles of the other iron, the bridging dithiolate, and the CO/CN ligands remain undefined.
          Proposed research: Gas phase simulations with simplified model complexes have already been performed for the active-site of the Fe-only hydrogenase. Using these published results as reference, an integrated molecular orbital/molecular mechanical approach51 will be applied to simulate the complete protein structure with the aim of defining electron-/proton-transfer pathways and reactant/product channels. Since the Fe-only hydrogenases contain numerous Fe-S clusters other than the H-cluster, the straightforward application of XAS to define the experimental ground-state of the active-site is not possible. Based on high (>90%) sequence homology of the hydrogenase subdomains and ferredoxins, it is promising to attempt the substraction of the ferredoxin XAS spectra from that of the Fe-only hydrogenase, and quantitate the difference as originating from the active-site. Structurally analogous model compounds of the H-cluster containing two iron centers, bridging thiolates and CO/CN ligands are available in literature and can be readily synthesized. These complexes will be studied by Fe and S K-edge XAS and DFT calculations to define experimental ground-state bonding and to correlate it with reactivity. Less attention has been focused on the non-metal containing active-site. It is more difficult to probe such an active-site experimentally; however, it is easier computationally. Using MM/MO computational aproaches, the complete enzyme will be structurally characterized and the potential energy surface of hydrogen function will be described.
          C-cycle: The anaerobic metabolism of CO2 involves several reduction steps52 going through carbamate, imine, seconder- and primer-amine intermediates as summarized in Figure 4.
          In the first step, CO2 is reduced to a formyl group as part of a carbamate. This formyl group is transformed in a Schift base condensation to an imine, which is then stepwise reduced to a methyl group. At the final state of the reaction, the methyl group is transferred to a thiolate substrate, which is activated by a Ni containing coenzyme called F430.
Figure 4
  Although the mechanistic cycle53 is still not fully understood at the molecular level, the terminal step in methane generation has already been the subject of computational studies54,55.
          Proposed research: The proposal aims to extend the computational studies to the entire process. The active-site structures of the enzymes involved in the methanogenesis function will be studied by computations and experiments. Protein samples will be isolated or obtained from collaborators. One of the long term goal of this research is to develop a shorter pathway for the reduction process, which would be greatly preferred in industrial applications. As in the case of the N- and H-cycles, the proposed computations would be strongly correlated with spectroscopic data. Since the molecular structures are not as well defined as for the N- or H-cycle, the use of other spectroscopic techniques such as magnetic circular dichroism, and resonance Raman, would also be necessary in defining the active-site structures and intermediates.
 
 

Research questions:

  1. What is the role of the protein environment in biological N2 and H2 activations and why do the structurally analogue biomimetic complexes not show the same reactivity?
  2. How do the nitrogenase, hydrogenase, and methanogenic enzymes interact, and what is the molecular basis of proton-, electron- and energy-transport?
  3. What is the molecular mechanism of CO2 reduction to CH4 and is there a technologically more viable way of involving less enzymatic steps and more simple substrates?
 
 

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