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9 Pág. Centro de Biotecnología y Genómica de Plantas, The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe-9S-C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe-S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe-4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals., This work was supported, in whole or in part, by the Bill and Melinda Gates Foundation Grants OPP1143172 and INV-005889. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. W.H. and C.B. were supported by doctoral fellowships from AGAUR. X.J. was supported by a doctoral fellowship from Universidad Politécnica de Madrid.

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, P

The “FeMo cofactors” in biological nitrogenase play a decisive role in nitrogen reduction. Herein, a novel bionic Fe/Mo bimetallene was applied in photocatalytic nitrogen reduction. The surface coating Fe/Mo bimetallene of Bi2Mo0.3W0.7O6 (BMWO) nanocrystals could effectively promote the separation and transportation of photogenerated carriers by multi–electron redox reactions and deliver 2.8 times longer photo–carrier lifetime. Consequently, the nitrogen fixation activity of Fe/Mo bimetallene–coated BMWO nanocrystal photocatalyst was obviously enhanced (218.93 μmol g–1h−1), which was about 4.8 times that of unmodified BMWO nanocrystals. This work provides a novel approach to design bionic Fe/Mo bimetallene–modified inorganic semiconductor photocatalysts for nitrogen reduction.

Energetically accessible reversible unhooking of S2B or S2BH from Fe2, as an intrinsic property of FeMo-co, needs to be considered in the formulation of mechanisms for the reactions of nitrogenase.

Carbon monoxide (CO) is a well-known inhibitor of nitrogenase activity. Under turnover conditions, CO binds to FeMoco, the active site of Mo nitrogenase. Time-resolved IR measurements suggest an initial terminal CO at 1904 cm–1 that converts to a bridging CO at 1715 cm–1, and an X-ray structure shows that CO can displace one of the bridging belt sulfides of FeMoco. However, the CO-binding redox state(s) of FeMoco (En) and the role of the protein environment in stabilizing specific CO-bound intermediates remain elusive. In this work, we carry out an in-depth analysis of the CO–FeMoco interaction based on quantum chemical calculations addressing different aspects of the electronic structure. (1) The local electronic structure of the Fe–CO bond is studied through diamagnetically substituted FeMoco. (2) A cluster model of FeMoco within a polarizable continuum illustrates how CO binding may affect the spin-coupling between the metal centers. (3) A QM/MM model incorporates the explicit influence of the amino acid residues surrounding FeMoco in the MoFe protein. The QM/MM model predicts both a terminal and a bridging CO in the E1 redox state. The scaled calculated CO frequencies (1922 and 1716 cm–1, respectively) are in good agreement with the experimentally observed IR bands supporting CO binding to the E1 state. Alternatively, an E2 state QM/MM model, which has the same atomic structure as the CO-bound X-ray structure, features a semi-bridging CO with a scaled calculated frequency (1718 cm–1) similar to the bridging CO in the E1 model., The MoFe protein of wild-type nitrogenase has been shown to bind CO during enzymatic turnover. According to our QM/MM model, the protein environment plays a pivotal role during the binding event. A detailed analysis of the electronic structure reveals that CO modulates the spin-coupling between Fe centers and therefore an atypical set of broken-symmetry determinants has to be considered in the DFT calculations.

The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, has been of great interest for understanding the catalytic strategy utilized to reduce dinitrogen to ammonia under ambient temperatures and pressures. The reduction mechanism of nitrogenase is generally envisioned as involving multiple cycles of electron and proton transfers, with the known substrates requiring at least two cycles. Solvent kinetic isotope effect experiments, in which changes of reaction rates or product distribution are measured upon enrichment of solvent with heavy atom isotopes, have been valuable for deciphering the mechanism of complex enzymatic reactions involving proton or hydrogen transfer. We report the distribution of ethylene, dihydrogen, and methane isotopologue products measured from nitrogenase-catalyzed reductions of acetylene, protons, and cyanide, respectively, performed in varying levels of deuterium enrichment of the solvent. As has been noted previously, the total rate of product formation by nitrogenase is largely insensitive to the presence of D

Mo-nitrogenase catalyzes the challenging N

Nitrogenase is the only enzyme that can convert N

Additional working hypotheses for the mechanism of the enzyme nitrogenase: the active site uses several binding sites with different mechanistic roles.

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (COMany of the molecules that form the building blocks of life contain nitrogen. This element makes up most of the gas in the atmosphere, but in this form, it does not easily react, and most organisms cannot incorporate atmospheric nitrogen into biological molecules. To get around this problem, some species of bacteria produce an enzyme complex called nitrogenase that can transform nitrogen from the air into ammonia. This process is called nitrogen fixation, and it converts nitrogen into a form that can be used to sustain life. The nitrogenase complex is made up of two proteins: the MoFe protein, which contains the active site that binds nitrogen, turning it into ammonia; and the Fe protein, which drives the reaction. Besides the nitrogen fixation reaction, the Fe protein is involved in other biological processes, but it was not thought to bind directly to nitrogen, or to any of the other small molecules that the nitrogenase complex acts on. The Fe protein contains a cluster of iron and sulfur ions that is required to drive the nitrogen fixation reaction, but the role of this cluster in the other reactions performed by the Fe protein remains unclear. To better understand the role of this iron sulfur cluster, Buscagan, Kaiser and Rees used X-ray crystallography, a technique that can determine the structure of molecules. This approach revealed for the first time that when nitrogenase reacts with a small molecule called selenocyanate, the selenium in this molecule can replace the sulfur ions of the iron sulfur cluster in the Fe protein. Buscagan, Kaiser and Rees also demonstrated that the Fe protein could still incorporate selenium ions in the absence of the MoFe protein, which has traditionally been thought to provide the site essential for transforming small molecules. These results indicate that the iron sulfur cluster in the Fe protein may bind directly to small molecules that react with nitrogenase. In the future, these findings could lead to the development of new molecules that artificially produce ammonia from nitrogen, an important process for fertilizer manufacturing. In addition, the iron sulfur cluster found in the Fe protein is also present in many other proteins, so Buscagan, Kaiser and Rees’ experiments may shed light on the factors that control other biological reactions.

Inspired by mechanistic proposals for N

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo–electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

CO-bound forms of nitrogenase are N

The nitrogen reduction reaction (NRR) is a renewable alternative to the energy- and CO

Mo-dependent nitrogenase is a major contributor to global biological N(2) reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe(7)MoS(9)C-homocitrate) contains a carbide (C(4−)) centered within a trigonal prismatic CFe(6) core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with (13)C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. (13)C-carbide ENDOR of the S = 3/2 resting state (E(0)) is remarkable, with an extremely small isotropic hyperfine coupling constant, (C)a = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the (13)C carbide isotropic hyperfine-coupling constant essentially unchanged, (C)a = −1.30 MHz. This indicates that both the E(0) and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to (C)a from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E(0) to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe(6) core structures of E(0) and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide–Fe bonds.

As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active‐site FeMo‐cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer–Tropsch‐type chemistry). The C−C bond forming capabilities of nitrogenase suggest that multiple CO or CO‐derived ligands bind to the active site. Herein, we report a crystal structure with two CO ligands coordinated to the FeMo‐cofactor of the molybdenum nitrogenase at 1.33 Å resolution. In addition to the previously observed bridging CO ligand between Fe2 and Fe6 of the FeMo‐cofactor, a new ligand binding mode is revealed through a second CO ligand coordinated terminally to Fe6. While the relevance of this state to nitrogenase‐catalyzed reactions remains to be established, it highlights the privileged roles for Fe2 and Fe6 in ligand binding, with multiple coordination modes available depending on the ligand and reaction conditions., Besides reducing dinitrogen, nitrogenases reduce CO to hydrocarbons, mediating C−C bond formation between multiple CO‐derived ligands. To shed light on how multiple C‐based ligands bind to the active site, we report a 1.33 Å resolution crystal structure of two CO ligands coordinated to the FeMo‐cofactor of molybdenum nitrogenase (see picture). The second CO ligand is coordinated to Fe6 in a previously unreported ligand binding mode.

There is an evergrowing demand for environment-friendly processes to synthesize ammonia (NH

The nitrogenase active-site cofactor must accumulate 4e

The bond-valence method has been used for valence calculations of FeMo/V cofactors in FeMo/V proteins using 51 crystallographic data sets of FeMo/V proteins from the Protein Data Bank. The calculations show molybdenum(III) to be present in MoFe7S9C(Cys)(HHis)[R-(H)homocit] (where H4homocit is homocitric acid, HCys is cysteine and HHis is histidine) in FeMo cofactors, while vanadium(III) with a more reduced iron complement is obtained for FeV cofactors. Using an error analysis of the calculated valences, it was found that in FeMo cofactors Fe1, Fe6 and Fe7 can be unambiguously assigned as iron(III), while Fe2, Fe3, Fe4 and Fe5 show different degrees of mixed valences for the individual Fe atoms. For the FeV cofactors in PDB entry 5n6y, Fe4, Fe5 and Fe6 correspond to iron(II), iron(II) and iron(III), respectively, while Fe1, Fe2, Fe3 and Fe7 exhibit strongly mixed valences. Special situations such as CO-bound and selenium-substituted FeMo cofactors and O(N)H-bridged FeV cofactors are also discussed and suggest rearrangement of the electron configuration on the substitution of the bridging S atoms.

We have made a systematic combined quantum mechanical and molecular mechanical (QM/MM) investigation of possible structures of the N2 bound state of nitrogenase. We assume that N2 is immediately protonated to a N2H2 state, thereby avoiding the problem of determining the position of the protons in the cluster. We have systematically studied both end-on and side-on structures, as well as both HNNH and NNH2 states. Our results indicate that the binding of N2H2 is determined more by interactions and steric clashes with the surrounding protein than by the intrinsic preferences of the ligand and the cluster. The best binding mode with both the TPSS and B3LYP density-functional theory methods has trans-HNNH terminally bound to Fe2. It is stabilised by stacking of the substrate with His-195 and Ser-278. However, several other structures come rather close in energy (within 3–35 kJ/mol) at least in some calculations: The corresponding cis-HNNH structure terminally bound to Fe2 is second best with B3LYP. A structure with HNNH2 terminally bound to Fe6 is second most stable with TPSS (where the third proton is transferred to the substrate from the homocitrate ligand). Structures with trans-HNNH, bound to Fe4 or Fe6, or cis-HNNH bound to Fe6 are also rather stable. Finally, with the TPSS functional, a structure with cis-HNNH side-on binding to the Fe3–Fe4–Fe5–Fe7 face of the cluster is also rather low in energy, but all side-on structures are strongly disfavoured by the B3LYP method.

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth., The work performed on the laboratory of DRD was supported by Bill and Melinda Gates Foundation Grants BNF Cereals Phase II (OPP1143172) and BNF Cereals Phase III (INV-005889). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.

We have used combined quantum mechanical and molecular mechanical (QM/MM) calculations to study the reaction mechanism of nitrogenase, assuming that none of the sulfide ligands dissociates. To avoid the problem that there is no consensus regarding the structure and protonation of the E

The nitrogenase (N2ase) enzyme family is responsible for the conversion of dinitrogen into biologically accessible ammonia, a critical step in the global nitrogen cycle. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction, but it can also be reduced to hydrocarbons catalyzed by all three N2ases, namely the wild-type Mo enzyme and select variants and the V and Fe nitrogenases, both of which are orders of magnitude more effective. CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, we investigated the interaction of CO with the α-R277H variant of the Azotobacter vinelandii N2ase MoFe protein, in which the α-subunit 277Arg residue is replaced by His and results in production of only the S = 3/2 EPR signal (denoted as hi(5)-CO). Fourier-transform infrared (FT-IR) spectroscopy was used to follow the photolysis of CO bound to the α-R277H variant under cryogenic conditions. Multiple EPR-silent species were observed with FT-IR spectroscopic signatures previously assigned to CO-inhibited forms of the α-H195Q and α-H195N N2ase variants. The distribution of these CO-inhibited forms varied dramatically with pH over the range of pH 6.5 to pH 8.5, indicating protonation/deprotonation involvement.

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe(7)S(8)C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe(3)S(3)C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M’Fe(3)S(3)X](n−), where M’ = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M’ results in minor changes, the chelating, μ(3)-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Nitrogenase is a versatile metalloenzyme that reduces N

International audience; Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglianeuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Deutsche Forschungsgemeinschaft RTG1976, The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants., Work performed in the laboratory of D.R.D. was supported by Bill and Melinda Gates Foundation grants BNF Cereals Phase II (OPP1143172) and BNF Cereals phase III (INV-005889), work performed in the laboratory of O.E. was supported by funds from Deutsche Forschungsgemeinschaft grant RTG1976, and work performed in the laboratory of L.C.S. was supported by a grant from the U.S. Department of Energy, Office of Science, (BES) DE-SC0010687.

The iron-molybdenum cofactor (FeMoco) is responsible for dinitrogen reduction in Mo nitrogenase. Unlike the resting state, E

Nitrogenase catalyzes the reduction of N2 to NH3, supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin (in vivo) and sodium dithionite (in vitro). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitrogenase. Although it is common to use methyl viologen as an electron donor for many low-potential oxidoreductases, decreased nitrogenase activity is observed with an increasing concentration of methyl viologen, limiting its utility under many circumstances. In this work, we suggest that this concentration-dependent decrease in activity can be explained by the formation of a dimer of the radical cation of methyl viologen (Me2V•+)2 at higher methyl viologen concentrations. In addition, viologens functionalized with positively and negatively charged groups were synthesized and studied using spectroscopy and cyclic voltammetry. A sulfonated viologen derivative, 1,1′-bis(3-sulfonatopropyl)-4,4′-bipyridinium radical {[(SPr)2V•]−}, was found to support full nitrogenase activity up to a mediator concentration of 3 mM, while the positively charged viologen derivative was not an efficient reductant of nitrogenase due to the high standard redox potential. The utility of [(SPr)2V•]− as an electron donor for nitrogenase was demonstrated by a simple, sensitive spectrophotometric assay for nitrogenase activity that can provide accurate values for the specific activity and turnover rate constant under argon. Under N2, the formation of ammonia was confirmed. Because of the observed full activity of nitrogenase and low overpotential, [(SPr)2V•]− should also prove to be valuable for nitrogenase electrocatalysis, including bioelectrosynthetic N2 reduction.

The size and complexity of Mo-dependent nitrogenase, a multicomponent enzyme capable of reducing dinitrogen to ammonia, have made a detailed understanding of the FeMo cofactor (FeMoco) active site electronic structure an ongoing challenge. Selective substitution of sulfur by selenium in FeMoco affords a unique probe wherein local Fe-Se interactions can be directly interrogated via high-energy resolution fluorescence detected X-ray absorption spectroscopic (HERFD XAS) and extended X-ray absorption fine structure (EXAFS) studies. These studies reveal a significant asymmetry in the electronic distribution of the FeMoco, suggesting a more localized electronic structure picture than is typically assumed for iron-sulfur clusters. Supported by experimental small molecule model data in combination with time dependent density functional theory (TDDFT) calculations, the HERFD XAS data is consistent with an assignment of Fe2/Fe6 as an antiferromagnetically coupled diferric pair. HERFD XAS and EXAFS have also been applied to Se-substituted CO-inhibited MoFe protein, demonstrating the ability of these methods to reveal electronic and structural changes that occur upon substrate binding. These results emphasize the utility of Se HERFD XAS and EXAFS for selectively probing the local electronic and geometric structure of FeMoco.

Episodic future thinking depends on a core network of regions that involves, in addition to the medial temporal lobes (MTL), the ventromedial prefrontal cortex (vmPFC). Neuroimaging studies suggest that vmPFC is particularly involved when future thinking requires consideration of self-relevant information, but lesion evidence for a special role of vmPFC in constructing self-relevant scenarios is limited. To clarify the involvement of vmPFC in future thinking, eight patients with vmPFC lesions were asked to imagine future events pertaining to the self or to another person, and their performance was contrasted with that of eight patients with MTL lesions. Patients with vmPFC lesions were no more detailed in their description of future events pertaining to the self than of events pertaining to another person. In contrast, like controls, patients with MTL lesions showed a self-benefit, despite impoverished performance overall. These findings accord with evidence from neuroimaging studies and elucidate the distinct contributions of vmPFC and MTL to future thinking.

Nitrogenases utilize Fe–S clusters to reduce N(2) to NH(3). The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating (57)Fe into specific sites of nitrogenase cofactors, and we report herein site-selective (57)Fe labeling of the L-cluster—a carbide-containing, [Fe(8)S(9)C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with (57)Fe(2+) results in (57)Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with (57)Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E(4)(4H)) with two [Fe─H─Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E(4)(4H) hydrides showed that enzymatic re of E(4)(4H) hydride yields an H(2)-bound complex (E(4)(H(2),2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe─H bonds to the metal ions themselves enables N(2) to bind with concomitant H(2) release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E(4)(H(2),2H) has not been trapped, cryogenic photolysis of E(4)(4H) provides a means to address this question. Photolysis of E(4)(4H) causes hydnde-re with release of H(2), generating doubly reduced FeMo-co (denoted E(4)(2H)*), the extreme limit of the electron-density redistribution upon formation of E(4)(H(2),2H). Here we examine the doubly reduced FeMo-co core of the E(4)(2H)* limiting-state by (1)H, (57)Fe, and (95)Mo ENDOR to illuminate the partial electron-density redistribution upon E(4)(H(2),2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E(4)(2H)* ENDOR results as extended by DFT computations include (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; and (ii) two distinctive E(4)(4H) (57)Fe signals are suggested as associated with structurally identified “anchors” of one bridging hydride, two others with identified anchors of the second, with NBO-analysis further identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe─H bonds.

OBJECTIVE Proximity of brainstem cavernous malformations (BSCMs) to tracts and cranial nerve nuclei make it costly to transgress normal tissue in accessing the lesion or disrupting normal tissue adjacent to the lesion in the separation plane. This interplay between tissue sensitivity and extreme eloquence makes it difficult to avoid leaving a remnant on occasion. Recurrences require operative intervention, which may increase morbidity, lengthen recovery, and add to overall costs. An approximately 20-year experience with patients with recurrent BSCM lesions following primary microsurgical resection was reviewed. METHODS A prospectively maintained database of 802 patients who underwent microsurgical resection of cerebral cavernous malformations during 1997–2018 was queried to identify 213 patients with BSCMs. A retrospective chart review was conducted for patients with recurrent BSCM after primary resection who required a second surgery. RESULTS Fourteen of 213 patients (6.6%) underwent repeat resection for recurrent BSCM. Thirty-four hemorrhagic events were observed among these 14 patients over 576 patient-years (recurrent hemorrhage rate, 5.9% per year; median discrete hemorrhagic events, 2; median time to rehemorrhage, 897 days). BSCM occurred in the pons in 10 cases, midbrain in 2 cases, and medulla in 2 cases. A blind spot in the operative corridor was the most common cause of residual BSCM (9 patients). All recurrent BSCMs were removed completely, although 2 patients each required 2 operations to treat recurrence. Twelve patients had unchanged or improved modified Rankin Scale scores at last clinical evaluation compared with admission, and 2 patients had worse scores. Recurrence was more common among patients who were operated on in the first versus the second half of the series (8.5% vs 4.7%). CONCLUSIONS The 6.6% rate of BSCM recurrence requiring reoperation reflects the fine lines between complete resection and recurrence and between safe and harmful surgery. The detection of remnants is difficult postoperatively and remains so even at 6 months when the resection bed has healed. The 5.9% annual hemorrhage risk associated with recurrent BSCM in this experience is consistent with that reported for unoperated BSCMs. The right-angle method helps to anticipate blind spots and meticulously inspect the resection cavity for residual BSCM during surgery. A low percentage of recurrent BSCM (5%–10%) ensures ongoing effort toward an acceptable balance of safety and completeness.