Putative reaction mechanism of nitrogenase after dissociation of a sulfide ligand

We have investigated the implications of the recent crystallographic findings that the m2-bridging S2B sulfide ligand may reversibly dissociate from the active-site FeMo cluster of nitrogenase. We show with combined quantum mechanical and molecular mechanical (QM/MM) calculations that once S2B has dis- sociated, N2 may bind in that position and can be protonated to two NH3 groups by thermodynamically favourable steps. The substrate forms hydrogen bonds with two protein ligands, Gln-191 and His-195. For all steps, we have studied three possible protonation states of His-195 (protonated on either ND1, NE2 or both). We find that the thermodynamically favoured path involves an end-on NNH2 structure, a mixed side-on/end-on H2NNH structure, a side-on H2NNH2 structure, a bridging NH2 structure and a bridging NH3 structure. In all cases, His-195 seems to be protonated on the NE2 atom. Dissociation of the NH3 pro- duct is often unfavourable and requires either further reduction or protonation of the cluster or rebinding of S2B. In conclusion, our calculations show that dissociation of S2B gives rise to a natural binding and reaction site for nitrogenase, between the Fe2 and Fe6 atoms, which can support an alternating reaction mechanism with favourable energetics

What Is the Structure of the E4 Intermediate in Nitrogenase?

Nitrogenase is the only enzyme that can cleave the strong triple bond inN2. The active site contains a complicated MoFe7S9C cluster. It is believed that itneeds to accept four protons and electrons, forming the E4 state, before it can bind N2.However, there is no consensus on the atomic structure of the E4 state. Experimentalstudies indicate that it should contain two hydride ions bridging two pairs of Fe ions,and it has been suggested that both hydride ions as well as the two protons bind on thesame face of the cluster. On the other hand, density functional theory (DFT) studieshave indicated that it is energetically more favorable with either three hydride ions or with a triply protonated carbide ion, depending on the DFT functional. We have performed a systematic combined quantum mechanical and molecular mechanical (QM/MM) study of possible E4 states with two bridging hydride ions. Our calculations suggest that the most favorable structure has hydride ions bridging the Fe2/6 and Fe3/7 ion pairs. In fact, such structures are 14 kJ/mol more stable than structures with three hydride ions, showing that pure DFT functionals give energetically most favorable structures in agreement with experiments. An important reason for this finding is that we have identified a new type of broken-symmetry state that involves only two Fe ions with minority spin, in contrast to the previously studied states with three Fe ions with minority spin. The energetically best structures have the two hydride ions on different faces of the FeMo cluster, whereas better agreement with ENDOR data is obtained if they are on the same face; such structures are only 6−22 kJ/mol less stable

Quantum Mechanics/Molecular Mechanics Study of Resting-State Vanadium Nitrogenase: Molecular and Electronic Structure of the Iron–Vanadium Cofactor

Publisher's version (útgefin grein)The nitrogenase enzymes are responsible for all biological nitrogen reduction. How this is accomplished at the atomic level, however, has still not been established. The molybdenum-dependent nitrogenase has been extensively studied and is the most active catalyst for dinitrogen reduction of the nitrogenase enzymes. The vanadium-dependent form, on the other hand, displays different reactivity, being capable of CO and CO2 reduction to hydrocarbons. Only recently did a crystal structure of the VFe protein of vanadium nitrogenase become available, paving the way for detailed theoretical studies of the iron-vanadium cofactor (FeVco) within the protein matrix. The crystal structure revealed a bridging 4-atom ligand between two Fe atoms, proposed to be either a CO32- or NO3- ligand. Using a quantum mechanics/molecular mechanics model of the VFe protein, starting from the 1.35 Å crystal structure, we have systematically explored multiple computational models for FeVco, considering either a CO32- or NO3- ligand, three different redox states, and multiple broken-symmetry states. We find that only a [VFe7S8C(CO3)]2- model for FeVco reproduces the crystal structure of FeVco well, as seen in a comparison of the Fe-Fe and V-Fe distances in the computed models. Furthermore, a broken-symmetry solution with Fe2, Fe3, and Fe5 spin-down (BS7-235) is energetically preferred. The electronic structure of the [VFe7S8C(CO3)]2- BS7-235 model is compared to our [MoFe7S9C]- BS7-235 model of FeMoco via localized orbital analysis and is discussed in terms of local oxidation states and different degrees of delocalization. As previously found from Fe X-ray absorption spectroscopy studies, the Fe part of FeVco is reduced compared to FeMoco, and the calculations reveal Fe5 as locally ferrous. This suggests resting-state FeVco to be analogous to an unprotonated E1 state of FeMoco. Furthermore, V-Fe interactions in FeVco are not as strong compared to Mo-Fe interactions in FeMoco. These clear differences in the electronic structures of otherwise similar cofactors suggest an explanation for distinct differences in reactivity.R.B. acknowledges support from the Icelandic Research Fund (Grants 141218051 and 162880051) and University of Iceland Research Fund. Open Access funding was provided by the Max Planck Society.Peer Reviewe

Implications of Metal Coordination in Damage and Recognition of Nucleic Acids and Lipid Bilayers

Metal ions have a myriad of biological functions from structural stability to enzymatic (de)activation and metabolic electron transfer. Redox-active metals also mediate the formation of reactive oxygen species which may either cause oxidative damage or protect cellular components. Computational modeling is used here to investigate the role of (1) metal-ion binding to antimicrobial peptides, (2) metal-ion removal and disulfide formation on zinc finger (ZF) proteins, and (3) coordination of thiones/selones for the prevention of metal-mediated redox damage. Piscidins, natural-occurring antimicrobial peptides, efficiently kill bacteria by targeting their membranes. Their efficacy is enhanced in vitro by metal-binding and the presence of membrane-destabilizing oxidized phospholipids (oxPLs). Molecular dynamics (MD) simulations are used to model insertion of Ni2+-bound piscidins 1 (P1:Ni2+) and 3 (P3:Ni2+) into lipid bilayers in the presence and absence of oxPLs. Metallation promotes deeper peptide insertion in the membrane, and P1:Ni2+ is suggested to interact more with anionic lipid headgroups in the presence of oxPLs. The release of Zn2+ from ZF proteins through oxidation of the cysteine thiolates is associated with inhibition of viral replication, disruption of cancer gene expression, and DNA repair preventing tumor growth. Multi-microsecond MD simulations were performed to examine the effect of cysteine oxidation on the ZF456 fragment of transcription factor IIIA and its complex with 5S RNA. Upon oxidation in the absence of RNA, the individual ZF domains unfold yielding a globular ZF456 peptide. Oxidation of the RNA-bound ZF456 peptide disrupts key hydrogen bonding interactions between ZF5/ZF6 and 5S RNA. The antioxidant properties of sulfur and selenium compounds prevent metal-mediated (i.e., Fenton chemistry) oxidative damage. The effect of the coordination of sulfur/selenium derivatives of imidazolidinone (thiones/selones) on the electronic structure and reduction potential of Fe2+ ions solvated or coordinated to guanine are examined using density functional theory. The highest occupied molecular orbital (HOMO) for the Fe(II)-aqua complex is metal-centered but localized on the nucleobase in the Fe2+-guanine complex. Complexation of the thione/selone shifts the HOMO to the sulfur/selenium center suggesting a mechanism for protection of DNA by sacrificial oxidation of the sulfur/selenium ligand

Old dog, new tricks : repurposing iron-carbide-carbonyl clusters as precursors for structural modeling of the nitrogenase cofactor

Nitrogenases are the only known biological enzyme capable of catalyzing the transformation of dinitrogen (N₂) into ammonia (NH₃). The active site of nitrogenase is comprised of a double-cuboidal iron-sulfur cluster featuring an interstitial carbide as the shared vertex, three ‘belt’ sulfides bridging the cuboidal components, and either a homocitrate-bearing heterometal (Mo, V) or an Fe at one of the distal capping metal sites. Out of the three nitrogenases, the Mo-dependent variant demonstrates the highest activity for N₂ conversion. The active-site cofactor of Mo-dependent nitrogenase (FeMoco) was first isolated in 1977; however, after decades of kinetic, structural, and spectroscopic research, many questions surrounding the mechanism of substrate reduction and the electronic structure of reaction intermediates remain unanswered. In this regard, the synthetic modelling community has contributed significantly towards directing mechanistic discussions with N₂-reducing functional model compounds. Furthermore, structural model compounds have played a pivotal role in deciphering the structural and electronic properties of FeMoco, including the identification of the central carbide and assignment of metal-site valence and spin states. Despite this remarkable progress, a synthetic model featuring a paramagnetic iron cluster with sulfides, interstitial carbide, and heterometal Mo has yet to be reported. The work relayed in this dissertation outlines our efforts towards pursuing this synthetic goal. As such, we utilize a family of carbonyl-supported iron clusters — first reported in the 1960s — featuring iron-coordinated inorganic carbide. However, the highly symmetric packing structures have made heterometal-containing carbidocarbonyl iron clusters difficult to unambiguously characterize by X-ray crystallography. Moreover, the strongly π-acidic ligation sphere enforces low metal-valance states and overall diamagnetism, and ligand substitution of COs is difficult to control. Here, we demonstrate a strategy to disrupt the symmetry in molybdenum-containing heteroclusters to unambiguously characterize the Mo site in XRD. Additionally, CO→S ligand substitution is achieved with the utilization of electrophilic sulfur sources, leading to progressively higher oxidation state Fe sites. These synthetic approaches for heterometal incorporation and oxidative sulfur insertion will serve as fundamental stepping-stones towards future endeavors in utilizing and functionalizing carbidocarbonyl iron clusters as synthetic precursors and ultimately, in biomimetically modeling the nitrogenase active site cluster.Chemistr