Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase.

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.

Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase.

The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.

Hydroxyl Transfer to Carbon Radicals by Mn(OH) vs Fe(OH) Corrole Complexes.

The transfer of •OH from metal-hydroxo species to carbon radicals (R•) to give hydroxylated products (ROH) is a fundamental process in metal-mediated heme and nonheme C-H bond oxidations. This step, often referred to as the hydroxyl "rebound" step, is typically very fast, making direct study of this process challenging if not impossible. In this report, we describe the reactions of the synthetic models M(OH)(ttppc) (M = Fe (1), Mn (3); ttppc = 5,10,15-tris(2,4,6-triphenyl)phenyl corrolato3-) with a series of triphenylmethyl carbon radical (R•) derivatives ((4-X-C6H4)3C•; X = OMe, tBu, Ph, Cl, CN) to give the one-electron reduced MIII(ttppc) complexes and ROH products. Rate constants for 3 for the different radicals ranged from 11.4(1) to 58.4(2) M-1 s-1, as compared to those for 1, which fall between 0.74(2) and 357(4) M-1 s-1. Linear correlations for Hammett and Marcus plots for both Mn and Fe were observed, and the small magnitudes of the slopes for both correlations imply a concerted •OH transfer reaction for both metals. Eyring analyses of reactions for 1 and 3 with (4-X-C6H4)3C• (X = tBu, CN) also give good linear correlations, and a comparison of the resulting activation parameters highlight the importance of entropy in these •OH transfer reactions. Density functional theory calculations of the reaction profiles show a concerted process with one transition state for all radicals investigated and help to explain the electronic features of the OH rebound process.

Hydrogen Atom Abstraction by High-Valent Fe(OH) versus Mn(OH) Porphyrinoid Complexes: Mechanistic Insights from Experimental and Computational Studies.

High-valent metal-hydroxide species have been implicated as key intermediates in hydroxylation chemistry catalyzed by heme monooxygenases such as the cytochrome P450s. However, in some classes of P450s, a bifurcation from the typical oxygen rebound pathway is observed, wherein the FeIV(OH)(porphyrin) species carries out a net hydrogen atom transfer reaction to form alkene metabolites. In this work, we examine the hydrogen atom transfer (HAT) reactivity of FeIV(OH)(ttppc) (1), ttppc = 5,10,15-tris(2,4,6-triphenyl)-phenyl corrole, toward substituted phenol derivatives. The iron hydroxide complex 1 reacts with a series of para-substituted 2,6-di-tert-butylphenol derivatives (4-X-2,6-DTBP; X = OMe, Me, Et, H, Ac), with second-order rate constants k2 = 3.6(1)-1.21(3) × 104 M-1 s-1 and yielding linear Hammett and Marcus plot correlations. It is concluded that the rate-determining step for O-H cleavage occurs through a concerted HAT mechanism, based on mechanistic analyses that include a KIE = 2.9(1) and DFT calculations. Comparison of the HAT reactivity of 1 to the analogous Mn complex, MnIV(OH)(ttppc), where only the central metal ion is different, indicates a faster HAT reaction and a steeper Hammett slope for 1. The O-H bond dissociation energy (BDE) of the MIII(HO-H) complexes were estimated from a kinetic analysis to be 85 and 89 kcal mol-1 for Mn and Fe, respectively. These estimated BDEs are closely reproduced by DFT calculations and are discussed in the context of how they influence the overall H atom transfer reactivity.

Biomimetic Reactivity of Oxygen-Derived Manganese and Iron Porphyrinoid Complexes.

Heme proteins utilize the heme cofactor, an iron porphyrin, to perform a diverse range of reactions including dioxygen binding and transport, electron transfer, and oxidation/oxygenations. These reactions share several key metalloporphyrin intermediates, typically derived from dioxygen and its congeners such as hydrogen peroxide. These species are composed of metal-dioxygen, metal-superoxo, metal-peroxo, and metal-oxo adducts. A wide variety of synthetic metalloporphyrinoid complexes have been synthesized to generate and stabilize these intermediates. These complexes have been studied to determine the spectroscopic features, structures, and reactivities of such species in controlled and well-defined environments. In this Review, we summarize recent findings on the reactivity of these species with common porphyrinoid scaffolds employed for biomimetic studies. The proposed mechanisms of action are emphasized. This Review is organized by structural type of metal-oxygen intermediate and broken into subsections based on the metal (manganese and iron) and porphyrinoid ligand (porphyrin, corrole, and corrolazine).

A Reactive Manganese(IV)-Hydroxide Complex: A Missing Intermediate in Hydrogen Atom Transfer by High-Valent Metal-Oxo Porphyrinoid Compounds.

High-valent metal-hydroxide species are invoked as critical intermediates in both catalytic, metal-mediated O2 activation (e.g., by Fe porphyrin in Cytochrome P450) and O2 production (e.g., by the Mn cluster in Photosystem II). However, well-characterized mononuclear MIV(OH) complexes remain a rarity. Herein we describe the synthesis of MnIV(OH)(ttppc) (3) (ttppc = tris(2,4,6-triphenylphenyl) corrole), which has been characterized by X-ray diffraction (XRD). The large steric encumbrance of the ttppc ligand allowed for isolation of 3. The complexes MnV(O)(ttppc) (4) and MnIII(H2O)(ttppc) (1·H2O) were also synthesized and structurally characterized, providing a series of Mn complexes related only by the transfer of hydrogen atoms. Both 3 and 4 abstract an H atom from the O-H bond of 2,4-di- tert-butylphenol (2,4-DTBP) to give a radical coupling product in good yield (3 = 90(2)%, 4 = 91(5)%). Complex 3 reacts with 2,4-DTBP with a rate constant of k2 = 2.73(12) × 104 M-1 s-1, which is ∼3 orders of magnitude larger than 4 ( k2 = 17.4(1) M-1 s-1). Reaction of 3 with a series of para-substituted 2,6-di- tert-butylphenol derivatives (4-X-2,6-DTBP; X = OMe, Me, tBu, H) gives rate constants in the range k2 = 510(10)-36(1.4) M-1 s-1 and led to Hammett and Marcus plot correlations. Together with kinetic isotope effect measurements, it is concluded that O-H cleavage occurs by a concerted H atom transfer (HAT) mechanism and that the MnIV(OH) complex is a much more powerful H atom abstractor than the higher-valent MnV(O) complex, or even some FeIV(O) complexes.

Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex.

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process.

The Influence of Peripheral Substituent Modification on P(V), Mn(III), and Mn(V)(O) Corrolazines: X-ray Crystallography, Electrochemical and Spectroscopic Properties, and HAT and OAT Reactivities.

The influence of remote peripheral substitution on the physicochemical properties and reactivity of phosphorus and manganese corrolazine (Cz) complexes was examined. The substitution of p-MeO for p-t-Bu groups on the eight phenyl substituents of the ß-carbon atoms of the Cz ring led to changes in UV-vis transitions and redox potentials for each of the complexes. The oxygen atom transfer (OAT) and hydrogen atom transfer (HAT) reactivity of the Mn(V)(O) complexes was also influenced by p-MeO substitution. The OAT reactivity of Mn(V)(O)(MeOP8Cz) (MeOP8Cz = octakis(p-methoxyphenyl)corrolazinato(3-)) with triarylphosphine (PAr3) substrates led to second-order rate constants from 10.2(5) to 3.1(2) × 10(4) M(-1) s(-1). These rates of OAT are slower than those seen for Mn(V)(O)(TBP8Cz) (TBP8Cz = octakis(p-tert-butylphenyl)corrolazinato(3-)). A Hammett study involving para-substituted PAr3 substrates reveals a Hammett ρ-value for Mn(V)(O)(MeOP8Cz) that is more negative than that observed for Mn(V)(O)(TBP8Cz), consistent with a less electrophilic Mn center. The HAT reactivity of Mn(V)(O)(MeOP8Cz) with C-H substrates was examined and revealed second-order rate constants from 6.8(5) × 10(-5) to 1.70(2) × 10(-1) M(-1) s(-1). The rate constants varied with the C-H bond strength of the substrate. Slightly faster HAT rates with C-H substrates were observed with Mn(V)(O)(MeOP8Cz) compared to Mn(V)(O)(TBP8Cz), indicating that the basicity of the putative [Mn(IV)(O)](-) intermediate likely compensates for the more negative redox potential in the driving force for HAT. In addition, the complete, large-scale synthesis of the para-phenyl-substituted porphyrazines RP8PzH2 (R = p-tert-butylphenyl (TB), p-methoxyphenyl (MeO), and p-isopropylphenyl) and corrolazines RP8CzH3 (TBP8CzH3 and MeOP8CzH3) is presented. The crystal structures of the monoprotonated, metal-free corrolazine [(TBP8CzH3)(H)](+)[BArF](-), P(V)(OMe)2(MeOP8Cz), and Mn(III)(MeOP8Cz)(MeOH) are presented. This work provides the first insights into the influence of electronic substituent effects on the corrolazine periphery.

Strong Inhibition of O-Atom Transfer Reactivity for Mn(IV)(O)(π-Radical-Cation)(Lewis Acid) versus Mn(V)(O) Porphyrinoid Complexes.

The oxygen atom transfer (OAT) reactivity of two valence tautomers of a Mn(V)(O) porphyrinoid complex was compared. The OAT kinetics of Mn(V)(O)(TBP8Cz) (TBP8Cz = octakis(p-tert-butylphenyl)corrolazinato(3-)) reacting with a series of triarylphosphine (PAr3) substrates were monitored by stopped-flow UV-vis spectroscopy, and revealed second-order rate constants ranging from 16(1) to 1.43(6) × 10(4) M(-1) s(-1). Characterization of the OAT transition state analogues Mn(III)(OPPh3)(TBP8Cz) and Mn(III)(OP(o-tolyl)3)(TBP8Cz) was carried out by single-crystal X-ray diffraction (XRD). A valence tautomer of the closed-shell Mn(V)(O)(TBP8Cz) can be stabilized by the addition of Lewis and Brønsted acids, resulting in the open-shell Mn(IV)(O)(TBP8Cz(•+)):LA (LA = Zn(II), B(C6F5)3, H(+)) complexes. These Mn(IV)(O)(π-radical-cation) derivatives exhibit dramatically inhibited rates of OAT with the PAr3 substrates (k = 8.5(2) × 10(-3) - 8.7 M(-1) s(-1)), contrasting the previously observed rate increase of H-atom transfer (HAT) for Mn(IV)(O)(TBP8Cz(•+)):LA with phenols. A Hammett analysis showed that the OAT reactivity for Mn(IV)(O)(TBP8Cz(•+)):LA is influenced by the Lewis acid strength. Spectral redox titration of Mn(IV)(O)(TBP8Cz(•+)):Zn(II) gives Ered = 0.69 V vs SCE, which is nearly +700 mV above its valence tautomer Mn(V)(O)(TBP8Cz) (Ered = -0.05 V). These data suggest that the two-electron electrophilicity of the Mn(O) valence tautomers dominate OAT reactivity and do not follow the trend in one-electron redox potentials, which appear to dominate HAT reactivity. This study provides new fundamental insights regarding the relative OAT and HAT reactivity of valence tautomers such as M(V)(O)(porph) versus M(IV)(O)(porph(•+)) (M = Mn or Fe) found in heme enzymes.

Detecting and Characterizing the Kinetic Activation of Thermal Networks in Proteins: Thermal Transfer from a Distal, Solvent-Exposed Loop to the Active Site in Soybean Lipoxygenase.

The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317-334) that correlate well with trends in the corresponding enthalpic barriers for kcat. A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD (Ea(ksolv) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation (Ea(kcat) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced "protein quakes" as the origin of enthalpic barriers in catalysis.

meso-N-Methylation of a porphyrinoid complex: activating the H-atom transfer capability of an inert ReV(O) corrolazine.

The selective alkylation of a single meso-N atom of a corrolazine macrocycle is reported. Alkylation has a dramatic impact on the physicochemical properties of ReV(O)(TBP8Cz). New electron-transfer and hydrogen-atom-transfer reactivity is also seen for this complex, including one-electron reduction, which gives an air-stable 19π-electron aromatic radical complex.

Rhenium(V)-oxo corrolazines: isolating redox-active ligand reactivity.

The synthesis of the first example of a third-row metallocorrolazine characterized by single crystal X-ray diffraction is reported. This Re(V)(O) porphyrinoid complex shows an exclusively ligand-based reactivity with strong acids and oxidizing agents. The one-electron oxidized π-radical-cation complex is capable of H-atom abstraction.