Molecular Catalysts with Diphosphine Ligands Containing Pendant Amines.

Pendant amines play an invaluable role in chemical reactivity, especially for molecular catalysts based on earth-abundant metals. As inspired by [FeFe]-hydrogenases, which contain a pendant amine positioned for cooperative bifunctionality, synthetic catalysts have been developed to emulate this multifunctionality through incorporation of a pendant amine in the second coordination sphere. Cyclic diphosphine ligands containing two amines serve as the basis for a class of catalysts that have been extensively studied and used to demonstrate the impact of a pendant base. These 1,5-diaza-3,7-diphosphacyclooctanes, now often referred to as "P2N2" ligands, have profound effects on the reactivity of many catalysts. The resulting [Ni(PR2NR'2)2]2+ complexes are electrocatalysts for both the oxidation and production of H2. Achieving the optimal benefit of the pendant amine requires that it has suitable basicity and is properly positioned relative to the metal center. In addition to the catalytic efficacy demonstrated with [Ni(PR2NR'2)2]2+ complexes for the oxidation and production of H2, catalysts with diphosphine ligands containing pendant amines have also been demonstrated for several metals for many different reactions, both in solution and immobilized on surfaces. The impact of pendant amines in catalyst design continues to expand.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H2 formation.

Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e-/H+] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e-/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e-/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e-/H+] generates E4(4H) with Fe2-H-Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E2(2H) containing Fe3-H-Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E2(2H) contains Fe2-H-Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.

Multilevel Computational Studies Reveal the Importance of Axial Ligand for Oxygen Reduction Reaction on Fe-N-C Materials.

The systematic improvement of Fe-N-C materials for fuel cell applications has proven challenging, due in part to an incomplete atomistic understanding of the oxygen reduction reaction (ORR) under electrochemical conditions. Herein, a multilevel computational approach, which combines ab initio molecular dynamics simulations and constant potential density functional theory calculations, is used to assess proton-coupled electron transfer (PCET) processes and adsorption thermodynamics of key ORR intermediates. These calculations indicate that the potential-limiting step for ORR on Fe-N-C materials is the formation of the FeIII-OOH intermediate. They also show that an active site model with a water molecule axially ligated to the iron center throughout the catalytic cycle produces results that are consistent with the experimental measurements. In particular, reliable prediction of the ORR onset potential and the Fe(III/II) redox potential associated with the conversion of FeIII-OH to FeII and desorbed H2O requires an axial H2O co-adsorbed to the iron center. The observation of a five-coordinate rather than four-coordinate active site has significant implications for the thermodynamics and mechanism of ORR. These findings highlight the importance of solvent-substrate interactions and surface charge effects for understanding the PCET reaction mechanisms and transition-metal redox couples under realistic electrochemical conditions.

13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe-C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6 ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6 Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe-C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe-C bonding of the CFe6 core indicate that it does not provide a functionally dynamic (hemilabile) "beating heart"─instead it acts as "a heart of steel", stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

Mechanical coupling in the nitrogenase complex.

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue ß-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing ß-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing ß-188Ser) are important for communication between the two halves.

Weakening the N-H Bonds of NH3 Ligands: Triple Hydrogen-Atom Abstraction to Form a Chromium(V) Nitride.

Weakening and cleaving N-H bonds is crucial for improving molecular ammonia (NH3) oxidation catalysts. We report the synthesis and H-atom-abstraction reaction of bis(ammonia)chromium porphyrin complexes Cr(TPP)(NH3)2 and Cr(TMP)(NH3)2 (TPP = 5,10,15,20-tetraphenyl-meso-porphyrin and TMP = 5,10,15,20-tetramesityl-meso-porphyrin) using bulky aryloxyl radicals. The triple H-atom-abstraction reaction results in the formation of CrV(por)(≡N), with the nitride derived from NH3, as indicated by UV-vis and IR and single-crystal structural determination of Cr(TPP)(≡N). Subsequent oxidation of this chromium(V) nitrido complex results in the formation of CrIII(por), with scission of the Cr≡N bond. Computational analysis illustrates the progression from CrII to CrV and evaluates the energetics of abstracting H atoms from CrII-NH3 to generate CrV≡N. The formation and isolation of CrV(por)(≡N) illustrates the stability of these species and the need to chemically activate the nitride ligand for atom transfer or N-N coupling reactivity.

Hydrogen Atom Abstraction from an OsII(NH3)2 Complex Generates an OsIV(NH2)2 Complex: Experimental and Computational Analysis of the N-H Bond Dissociation Free Energies and Reactivity.

Double hydrogen atom abstraction from (TMP)OsII(NH3)2 (TMP = tetramesitylporphyrin) with phenoxyl or nitroxyl radicals leads to (TMP)OsIV(NH2)2. This unusual bis(amide) complex is diamagnetic and displays an N-H resonance at 12.0 ppm in its 1H NMR spectrum. 1H-15N correlation experiments identified a 15N NMR spectroscopic resonance signal at -267 ppm. Experimental reactivity studies and density functional theory calculations support relatively weak N-H bonds of 73.3 kcal/mol for (TMP)OsII(NH3)2 and 74.2 kcal/mol for (TMP)OsIII(NH3)(NH2). Cyclic voltammetry experiments provide an estimate of the pKa of [(TMP)OsIII(NH3)2]+. In the presence of Barton's base, a current enhancement is observed at the Os(III/II) couple, consistent with an ECE event. Spectroscopic experiments confirmed (TMP)OsIV(NH2)2 as the product of bulk electrolysis. Double hydrogen atom abstraction is influenced by π donation from the amides of (TMP)OsIV(NH2)2 into the d orbitals of the Os center, favoring the formation of (TMP)OsIV(NH2)2 over N-N coupling. This π donation leads to a Jahn-Teller distortion that splits the energy levels of the dxz and dyz orbitals of Os, results in a low-spin electron configuration, and leads to minimal aminyl character on the N atoms, rendering (TMP)OsIV(NH2)2 unreactive toward amide-amide coupling.

Reduction of Substrates by Nitrogenases.

Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to support food and chemical production and the heavy reliance of the industrial Haber-Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.

Protonation of Serine in Gas and Condensed and Microsolvated States in Aqueous Solution.

Identification of molecules and elucidation of their chemical structure are ubiquitous problems in chemistry. Mass spectrometry (MS) can be used due to its sensitivity and versatility. For detection to occur, analytes must be ionized and transferred to the gas phase. Soft ionization processes such as electrospray ionization are popular; however, resulting microsolvated phases can alter the chemistry of analytes and therefore detection and identification. To understand these processes, we use computational methods to probe the ionization propensity of serine in the gas phase, aqueous microsolvated clusters, and aqueous solution. We show that the tautomeric form of serine is altered by the presence of water, as five water molecules can stabilize the zwitterionic tautomer. Inclusion of cosolutes such as ions can stabilize the zwitterion with as few as one or two water molecules present. We demonstrate that ionization propensity, as measured by gas phase bacisity, can increase by over 100 kJ/mol when placed in a small water-serine cluster, showing the sensitivity of the chemistry of microsolvated analytes. Finally, detailed analysis reveals that small droplets (less than seven water molecules) are extremely sensitive to addition of further water molecules. Beyond this limit, structural and electronic properties change little with droplet size.

Atomistic insight on structure and dynamics of spinach acyl carrier protein with substrate length.

The plant acyl-acyl carrier protein (ACP) desaturases are a family of soluble enzymes that convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction. These enzymes play a key role in biosynthesis of monounsaturated fatty acids in plants. ACPs are central proteins in fatty acid biosynthesis that deliver acyl chains to desaturases. They have been reported to show a varying degree of local dynamics and structural variability depending on the acyl chain size. It has been suggested that substrate-specific changes in ACP structure and dynamics have a crucial impact on the desaturase enzymatic activity. Using molecular dynamics simulations, we investigated the intrinsic solution structure and dynamics of ACP from spinach with four different acyl chains: capric (C10), myristic (C14), palmitic (C16), and stearic (C18) acids. We found that the fatty acids can adopt two distinct structural binding motifs, which feature different binding free energies and influence the ACP dynamics in a different manner. Docking simulations of ACP to castor Δ9-desaturase and ivy Δ4-desaturase suggest that ACP desaturase interactions could lead to a preferential selection between the motifs.

Nickel-Sulfonate Mode of Substrate Binding for Forward and Reverse Reactions of Methyl-SCoM Reductase Suggest a Radical Mechanism Involving Long-Range Electron Transfer.

Methyl-coenzyme M reductase (MCR) catalyzes both the synthesis and the anaerobic oxidation of methane (AOM). Its catalytic site contains Ni at the core of cofactor F430. The Ni ion, in its low-valent Ni(I) state, lights the fuse leading to homolysis of the C-S bond of methyl-coenzyme M (methyl-SCoM) to generate a methyl radical, which abstracts a hydrogen atom from coenzyme B (HSCoB) to generate methane and the mixed disulfide CoMSSCoB. Direct reversal of this reaction activates methane to initiate anaerobic methane oxidation. On the basis of the crystal structures, which reveal a Ni-thiol interaction between Ni(II)-MCR and inhibitor CoMSH, a Ni(I)-thioether complex with substrate methyl-SCoM has been transposed to canonical MCR mechanisms. Similarly, a Ni(I)-disulfide with CoMSSCoB is proposed for the reverse reaction. However, this Ni(I)-sulfur interaction poses a conundrum for the proposed hydrogen-atom abstraction reaction because the >6 Å distance between the thiol group of SCoB and the thiol of SCoM observed in the structures appears to be too long for such a reaction. The spectroscopic, kinetic, structural, and computational studies described here establish that both methyl-SCoM and CoMSSCoB bind to the active Ni(I) state of MCR through their sulfonate groups, forming a hexacoordinate Ni(I)-N/O complex, not Ni(I)-S. These studies rule out direct Ni(I)-sulfur interactions in both substrate-bound states. As a solution to the mechanistic conundrum, we propose that both the forward and the reverse MCR reactions emanate through long-range electron transfer from the Ni(I)-sulfonate complexes with methyl-SCoM and CoMSSCoB, respectively.

Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N2 reduction.

Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N2 → 2NH3 reduction involves the reductive elimination (re) of H2 from two [Fe-H-Fe] bridging hydrides bound to the catalytic [7Fe-9S-Mo-C-homocitrate] FeMo-cofactor (FeMo-co). These studies rationalize the Lowe-Thorneley kinetic scheme's proposal of mechanistically obligatory formation of one H2 for each N2 reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i) This shows that to prevent spurious disruption of FeMo-co having accumulated 4[e-/H+] it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii) These calculations indicate an important role of sulfide hemilability in the overall conversion of E0 to a diazene-level intermediate. (iii) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H2 formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutral re/oxidative-addition equilibrium, (iiib) while preventing the "futile" generation of two H2 without N2 reduction: hydride re generates an H2 complex, but H2 is only lost when displaced by N2, to form an end-on N2 complex that proceeds to a diazene-level intermediate.

Selectivity-Determining Steps in O2 Reduction Catalyzed by Iron(tetramesitylporphyrin).

The oxygen reduction reaction (ORR) is the cathode reaction in fuel cells and its selectivity for water over hydrogen peroxide production is important for these technologies. Iron porphyrin catalysts have long been studied for the ORR, but the origins of their selectivity are not well understood because the selectivity-determining step(s) usually occur after the rate-determining step. We report here the effects of acid concentration, as well as other solution conditions such as acid pKa, on the H2O2/H2O selectivity in electrocatalytic ORR by iron(tetramesitylporphyrin) (Fe(TMP)) in DMF. The results show that selectivity reflects a kinetic competition in which the dependence on [HX] is one order greater for the production of H2O than H2O2. Based on such experimental results and computational studies, we propose that the selectivity is governed by competition between protonation of the hydroperoxo intermediate, FeIII(TMP)(OOH), to produce water versus dissociation of the HOO- ligand to yield H2O2. The data rule out a bifurcation based on the regioselectivity of protonation of the hydroperoxide, as suggested in the enzymatic systems. Furthermore, the analysis developed in this report should be generally valuable to the study of selectivity in other multi-proton/multi-electron electrocatalytic reactions.

Electron Redistribution within the Nitrogenase Active Site FeMo-Cofactor During Reductive Elimination of H2 to Achieve N≡N Triple-Bond Activation.

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E4(4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E4(4H) hydrides showed that enzymatic re of E4(4H) hydride yields an H2-bound complex (E4(H2,2H)), 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 N2 to bind with concomitant H2 release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E4(H2,2H) has not been trapped, cryogenic photolysis of E4(4H) provides a means to address this question. Photolysis of E4(4H) causes hydride-re with release of H2, generating doubly reduced FeMo-co (denoted E4(2H)*), the extreme limit of the electron-density redistribution upon formation of E4(H2,2H). Here we examine the doubly reduced FeMo-co core of the E4(2H)* limiting-state by 1H, 57Fe, and 95Mo ENDOR to illuminate the partial electron-density redistribution upon E4(H2,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E4(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 E4(4H) 57Fe 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.

Tuning Catalytic Bias of Hydrogen Gas Producing Hydrogenases.

Hydrogenases display a wide range of catalytic rates and biases in reversible hydrogen gas oxidation catalysis. The interactions of the iron-sulfur-containing catalytic site with the local protein environment are thought to contribute to differences in catalytic reactivity, but this has not been demonstrated. The microbe Clostridium pasteurianum produces three [FeFe]-hydrogenases that differ in "catalytic bias" by exerting a disproportionate rate acceleration in one direction or the other that spans a remarkable 6 orders of magnitude. The combination of high-resolution structural work, biochemical analyses, and computational modeling indicates that protein secondary interactions directly influence the relative stabilization/destabilization of different oxidation states of the active site metal cluster. This selective stabilization or destabilization of oxidation states can preferentially promote hydrogen oxidation or proton reduction and represents a simple yet elegant model by which a protein catalytic site can confer catalytic bias.

Intramolecular Electrostatic Effects on O2, CO2, and Acetate Binding to a Cationic Iron Porphyrin.

Noncovalent electrostatic interactions are important in many biological and chemical reactions, especially those that involve charged intermediates. There has been a growing interest in using electrostatic ligand designs-placing charges in the second coordination sphere-to improve molecular reactivity, catalysis, and electrocatalysis. For instance, an iron porphyrin bearing four cationic ortho-trimethylanilinium groups, Fe(o-TMA), has been reported to be an exceptional electrocatalyst for both the carbon dioxide reduction reaction (CO2RR) and the oxygen reduction reaction (ORR). These reactions involve many different steps, and it is not evident which steps are affected by the four positive charges, or why. By comparing Fe(o-TMA) with the related iron-tetraphenylporphyrin, this work examines how covalently positioned charged groups affect substrate binding and other key pre-equilibria of both the ORR and CO2RR, specifically acetate, dioxygen, and carbon dioxide binding. This study is among the first to directly measure the effects of electrostatics on ligand-binding. The results show that adding electrostatic groups to a catalyst design often results in a complex interplay of multiple effects, including changes in pre-equilibria prior to substrate binding, combinations of through-space and inductive contributions, and effects of ionic strength and solution dielectric. The inverse half-order dependence of binding constant on ionic strength is proposed as a clear marker for an electrostatic effect. The conclusions provide guidance for the increasingly popular electrostatic ligand designs in catalysis and other reactivity.

Chokepoints in Mechanical Coupling Associated with Allosteric Proteins: The Pyruvate Kinase Example.

Although the critical role of allostery in controlling enzymatic processes is well appreciated, there is a current dearth in our understanding of its underlying mechanisms, including communication between binding sites. One potential key aspect of intersite communication is the mechanical coupling between residues in a protein. Here, we introduce a graph-based computational approach to investigate the mechanical coupling between distant parts of a protein, highlighting effective pathways via which protein motion can transfer energy between sites. In this method, each residue is treated as a node on a weighted, undirected graph, in which the edges are defined by locally correlated motions of those residues and weighted by the strength of the correlation. The method was validated against experimental data on allosteric regulation in the human liver pyruvate kinase as obtained from full-protein alanine-scanning mutagenesis (systematic mutation) studies, as well as computational data on two G-protein-coupled receptors. The method provides semiquantitative information on the regulatory importance of specific structural elements. It is shown that these elements are key for the mechanical coupling between distant parts of the protein by providing effective pathways for energy transfer. It is also shown that, although there are a multitude of energy transfer pathways between distant parts of a protein, these pathways share a few common nodes that represent effective "chokepoints" for the communication.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase.

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E4(4H).

We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the "Janus" intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of "anchor" Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a recently developed pulsed-ENDOR protocol ("PESTRE") to obtain absolute hyperfine interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its "anchoring" Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result is the freeze-trapped, lowest-energy Janus intermediate structure, E4(4H)(a).