Identifying the Rate-Limiting Elementary Steps of Nitrogen Fixation with Single-Site Fe Model Complexes

Biomimetic nitrogen fixation provides an attractive alternative for the century-old Haber−Bosch process; however, the performance of the currently available molecular biomimetic catalysts is very limited. In this work, we are aiming to understand the catalytic cycle of one of the most promising biomimetic complex families that can be the cornerstone of future computer-aided rational design of biomimetic complexes. We calculate the Gibbs free energy of all elementary reaction steps of homogeneous dinitrogen reduction to NH3 on singlesite iron complexes with EPPP tetradentate ligands (E = B, Si). We examine all possible mechanisms and identify the dominant pathways and the critical elementary steps that can be ratedetermining in the catalytic cycle of nitrogen fixation. We find that the catalytic mechanism depends on the applied ligand and that the distal pathway observed with E = B is the most favorable route regarding the catalytic performance. Our calculations also reveal the lack of thermodynamic driving force in the last steps of the catalytic cycle that can be responsible for the low catalytic activity of the studied biomimetic catalysts. Our results can serve as a starting point for the rational design of biomimetic complexes, which should focus on establishing a steadily decreasing Gibbs free energy profile, as suggested by the Sabatier principle

First principles calculation of the reaction rates for ligand binding to myoglobin: the cases of NO and CO

Ligand binding by proteins is among the most fundamental processes in nature. Among these processes the binding of small gas molecules, such as O2, CO and NO to heme proteins has traditionally received vivid interest, which was further boosted by their recently recognized significant role in gas sensing in the body. At the heart of the binding of these ligands to the heme group is the spin-forbidden reaction between high-spin iron(II) and the ligand yielding a low spin adduct. We use computational means to address the complete mechanism of CO and NO binding by myoglobin. As it involves several steps occurring on different time-scales, molecular dynamics simulations were performed to address the diffusion of the ligand through the enzyme, and DFT calculations in combination with statistical rate calculation to investigate the spin-forbidden reaction. The calculations yielded rate constants in qualitative agreement with experiment and revealed that the bottle-neck of NO and CO binding is different: for NO diffusion was found to be rate-limiting, while for CO the spin-forbidden step is the slowest

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP : Phosphocholine Cytidylyltransferase

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40 degrees C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line