Ferric Heme Superoxide Reductive Transformations to Ferric Heme (Hydro)Peroxide Species: Spectroscopic Characterization and Thermodynamic Implications for H-Atom Transfer (HAT).

A new end-on low-spin ferric heme peroxide, [(PIm )FeIII -(O22- )]- (PIm -P), and subsequently formed hydroperoxide species, [(PIm )FeIII -(OOH)] (PIm -HP) are generated utilizing the iron-porphyrinate PIm with its tethered axial base imidazolyl group. Measured thermodynamic parameters, the ferric heme superoxide [(PIm )FeIII -(O2⋅- )] (PIm -S) reduction potential (E°') and the PIm -HP pKa value, lead to the finding of the OO-H bond-dissociation free energy (BDFE) of PIm -HP as 69.5 kcal mol-1 using a thermodynamic square scheme and Bordwell relationship. The results are validated by the observed oxidizing ability of PIm -S via hydrogen-atom transfer (HAT) compared to that of the F8 superoxide complex, [(F8 )FeIII -(O2.- )] (S) (F8 =tetrakis(2,6-difluorophenyl)porphyrinate, without an internally appended axial base imidazolyl), as determined from reactivity comparison of superoxide complexes PIm -S and S with the hydroxylamine (O-H) substrates TEMPO-H and ABNO-H.

Heme-FeIII Superoxide, Peroxide and Hydroperoxide Thermodynamic Relationships: FeIII-O2•- Complex H-Atom Abstraction Reactivity.

Establishing redox and thermodynamic relationships between metal-ion-bound O2 and its reduced (and protonated) derivatives is critically important for a full understanding of (bio)chemical processes involving dioxygen processing. Here, a ferric heme peroxide complex, [(F8)FeIII-(O22-)]- (P) (F8 = tetrakis(2,6-difluorophenyl)porphyrinate), and a superoxide complex, [(F8)FeIII-(O2•-)] (S), are shown to be redox interconvertible. Using Cr(η-C6H6)2, an equilibrium state where S and P are present is established in tetrahydrofuran (THF) at -80 °C, allowing determination of the reduction potential of S as -1.17 V vs Fc+/0. P could be protonated with 2,6-lutidinium triflate, yielding the low-spin ferric hydroperoxide species, [(F8)FeIII-(OOH)] (HP). Partial conversion of HP back to P using a derivatized phosphazene base gave a P/HP equilibrium mixture, leading to the determination of pKa = 28.8 for HP (THF, -80 °C). With the measured reduction potential and pKa, the O-H bond dissociation free energy (BDFE) of hydroperoxide species HP was calculated to be 73.5 kcal/mol, employing the thermodynamic square scheme and Bordwell relationship. This calculated O-H BDFE of HP, in fact, lines up with an experimental demonstration of the oxidizing ability of S via hydrogen atom transfer (HAT) from TEMPO-H (2,2,6,6-tetramethylpiperdine-N-hydroxide, BDFE = 66.5 kcal/mol in THF), forming the hydroperoxide species HP and TEMPO radical. Kinetic studies carried out with TEMPO-H(D) reveal second-order behavior, kH = 0.5, kD = 0.08 M-1 s-1 (THF, -80 °C); thus, the hydrogen/deuterium kinetic isotope effect (KIE) = 6, consistent with H-atom abstraction by S being the rate-determining step. This appears to be the first case where experimentally derived thermodynamics lead to a ferric heme hydroperoxide OO-H BDFE determination, that FeIII-OOH species being formed via HAT reactivity of the partner ferric heme superoxide complex.

Geometric and Electronic Structure Contributions to O-O Cleavage and the Resultant Intermediate Generated in Heme-Copper Oxidases.

This study investigates the mechanism of O-O bond cleavage in heme-copper oxidase (HCO) enzymes, combining experimental and computational insights from enzyme intermediates and synthetic models. It is determined that HCOs undergo a proton-initiated O-O cleavage mechanism where a single water molecule in the active site enables proton transfer (PT) from the cross-linked tyrosine to a peroxo ligand bridging the heme FeIII and CuII, and multiple H-bonding interactions lower the tyrosine p Ka. Due to sterics within the active site, the proton must either transfer initially to the O(Fe) (a high-energy intermediate), or from another residue over a ∼10 Å distance to reach the O(Cu) atom directly. While the distance between the H+ donor (Tyr) and acceptor (O(Cu)) results in a barrier to PT, this separation is critical for the low barrier to O-O cleavage as it enhances backbonding from Fe into the O22- σ* orbital. Thus, PT from Tyr precedes O-O elongation and is rate-limiting, consistent with available kinetic data. The electron transfers from tyrosinate after the barrier via a superexchange pathway provided by the cross-link, generating intermediate PM. PM is evaluated using available experimental data. The geometric structure contains an FeIV═O that is H-bonded to the CuII-OH. The electronic structure is a singlet, where the FeIV and CuII are antiferromagnetically coupled through the H-bond between the oxo(Fe) and hydroxo(Cu) ligands, while the CuII and Tyr• are ferromagnetically coupled due their delocalization into orthogonal magnetic orbitals on the cross-linked His residue. These findings provide critical insights into the mechanism of efficient O2 reduction in HCOs, and the nature of the PM intermediate that couples this reaction to proton pumping.

Ligand Identity-Induced Generation of Enhanced Oxidative Hydrogen Atom Transfer Reactivity for a CuII2(O2•-) Complex Driven by Formation of a CuII2(-OOH) Compound with a Strong O-H Bond.

A superoxide-bridged dicopper(II) complex, [CuII2(XYLO)(O2•-)]2+ (1) (XYLO = binucleating m-xylyl derivative with a bridging phenolate ligand donor and two bis(2-{2-pyridyl}ethyl)amine arms), was generated from chemical oxidation of the peroxide-bridged dicopper(II) complex [CuII2(XYLO)(O22-)]+ (2), using ferrocenium (Fc+) derivatives, in 2-methyltetrahydrofuran (MeTHF) at -125 °C. Using Me10Fc+, a 1 ⇆ 2 equilibrium was established, allowing for calculation of the reduction potential of 1 as -0.525 ± 0.01 V vs Fc+/0. Addition of 1 equiv of strong acid to 2 afforded the hydroperoxide-bridged dicopper(II) species [CuII2(XYLO)(OOH)]2+ (3). An acid-base equilibrium between 3 and 2 was achieved through spectral titrations using a derivatized phosphazene base. The pKa of 3 was thus determined to be 24 ± 0.6 in MeTHF at -125 °C. Using a thermodynamic square scheme and the Bordwell relationship, the hydroperoxo complex (3) O-H bond dissociation free energy (BDFE) was calculated as 81.8 ± 1.5 (BDE = 86.8) kcal/mol. The observed oxidizing capability of [CuII2(XYLO)(O2•-)]2+ (1), as demonstrated in H atom abstraction reactions with certain phenolic ArO-H and hydrocarbon C-H substrates, provides direct support for this experimentally determined O-H BDFE. A kinetic study reveals a very fast reaction of TEMPO-H with 1 in MeTHF, with k (-100 °C) = 5.6 M-1 s-1. Density functional theory (DFT) calculations reveal how the structure of 1 may minimize stabilization of the superoxide moiety, resulting in its enhanced reactivity. The thermodynamic insights obtained herein highlight the importance of the interplay between ligand design and the generation and properties of copper (or other metal ion) bound O2-derived reduced species, such as pKa, reduction potential, and BDFE; these may be relevant to the capabilities (i.e., oxidizing power) of reactive oxygen intermediates in metalloenzyme chemical system mediated oxidative processes.

Spin Interconversion of Heme-Peroxo-Copper Complexes Facilitated by Intramolecular Hydrogen-Bonding Interactions.

Synthetic peroxo-bridged high-spin (HS) heme-(µ-η2:η1-O22-)-Cu(L) complexes incorporating (as part of the copper ligand) intramolecular hydrogen-bond (H-bond) capabilities and/or steric effects are herein demonstrated to affect the complex's electronic and geometric structure, notably impacting the spin state. An H-bonding interaction with the peroxo core favors a low-spin (LS) heme-(µ-η1:η1-O22-)-Cu(L) structure, resulting in a reversible temperature-dependent interconversion of spin state (5 coordinate HS to 6 coordinate LS). The LS state dominates at low temperatures, even in the absence of a strong trans-axial heme ligand. Lewis base addition inhibits the H-bond facilitated spin interconversion by competition for the H-bond donor, illustrating the precise H-bonding interaction required to induce spin-crossover (SCO). Resonance Raman spectroscopy (rR) shows that the H-bonding pendant interacts with the bridging peroxide ligand to stabilize the LS but not the HS state. The H-bond (to the Cu-bound O atom) acts to weaken the O-O bond and strengthen the Fe-O bond, exhibiting ν(M-O) and ν(O-O) values comparable to analogous known LS complexes with a strong donating trans-axial ligand, 1,5-dicyclohexylimidazole, (DCHIm)heme-(µ-η1:η1-O22-)-Cu(L). Variable-temperature (-90 to -130 °C) UV-vis and 2H NMR spectroscopies confirm the SCO process and implicate the involvement of solvent binding. Examining a case of solvent binding without SCO, thermodynamic parameters were obtained from a van't Hoff analysis, accounting for its contribution in SCO. Taken together, these data provide evidence for the H-bond group facilitating a core geometry change and allowing solvent to bind, stabilizing a LS state. The rR data, complemented by DFT analysis, reveal a stronger H-bonding interaction with the peroxo core in the LS compared to the HS complexes, which enthalpically favors the LS state. These insights enhance our fundamental understanding of secondary coordination sphere influences in metalloenzymes.

Heme-Cu Binucleating Ligand Supports Heme/O2 and FeII-CuI/O2 Reactivity Providing High- and Low-Spin FeIII-Peroxo-CuII Complexes.

The focus of this study is in the description of synthetic heme/copper/O2 chemistry employing a heme-containing binucleating ligand which provides a tridentate chelate for copper ion binding. The addition of O2 (-80 °C, tetrahydrofuran (THF) solvent) to the reduced heme compound (PImH)FeII (1), gives the oxy-heme adduct, formally a heme-superoxide complex FeIII-(O2•-) (2) (resonance Raman spectroscopy (rR): νO-O, 1171 cm-1 (Δ18O2, -61 cm-1); νFe-O, 575 cm-1 (Δ18O2, -24 cm-1)). Simple warming of 2 to room temperature regenerates reduced complex 1; this reaction is reversible, as followed by UV-vis spectroscopy. Complex 2 is electron paramagnetic resonance (EPR)-silent and exhibits upfield-shifted pyrrole resonances (δ 9.12 ppm) in 2H NMR spectroscopy, indicative of a six-coordinate low-spin heme. The coordination of the tethered imidazolyl arm to the heme-superoxide complex as an axial base ligand is suggested. We also report the new fully reduced heme-copper complex [(PImH)FeIICuI]+ (3), where the copper ion is bound to the tethered tridentate portion of PImH. This reacts with O2 to give a distinctive low-temperature-stable, high-spin (S = 2, overall) peroxo-bridged complex [(PImH)FeIII-(O22-)-CuII]+ (3a): λmax, 420 (Soret), 545, 565 nm; δpyrr, 93 ppm; νO-O, 799 cm-1 (Δ18O2, -48 cm-1); νFe-O, 524 cm-1 (Δ18O2, -23 cm-1). To 3a, the addition of dicyclohexylimidazole (DCHIm), which serves as a heme axial base, leads to low-spin (S = 0 overall) species complex [(DCHIm)(PImH)FeIII-(O22-)-CuII]+ (3b): λmax, 425 (Soret), 538 nm; δpyrr, 10.2 ppm; νO-O, 817 cm-1 (Δ18O2, -55 cm-1); νFe-O, 610 cm-1 (Δ18O2, -26 cm-1). These investigations into the characterization of the O2-adducts from (PImH)FeII (1) with/without additional copper chelation advance our understanding of the dioxygen reactivity of heme-only and heme/Cu-ligand heterobinuclear system, thus potentially relevant to O2 reduction in heme-copper oxidases or fuel-cell chemistry.

Impact of Intramolecular Hydrogen Bonding on the Reactivity of Cupric Superoxide Complexes with O-H and C-H Substrates.

The dioxygen reactivity of a series of TMPA-based copper(I) complexes (TMPA=tris(2-pyridylmethyl)amine), with and without secondary-coordination-sphere hydrogen-bonding moieties, was studied at -135 °C in 2-methyltetrahydrofuran (MeTHF). Kinetic stabilization of the H-bonded [( (X1)(X2) TMPA)CuII (O2.- )]+ cupric superoxide species was achieved, and they were characterized by resonance Raman (rR) spectroscopy. The structures and physical properties of [( (X1)(X2) TMPA)CuII (N3- )]+ azido analogues were compared, and the O2.- reactivity of ligand-CuI complexes when an H-bonding moiety is replaced by a methyl group was contrasted. A drastic enhancement in the reactivity of the cupric superoxide towards phenolic substrates as well as oxidation of substrates possessing moderate C-H bond-dissociation energies is observed, correlating with the number and strength of the H-bonding groups.

Phenol-Induced O-O Bond Cleavage in a Low-Spin Heme-Peroxo-Copper Complex: Implications for O2 Reduction in Heme-Copper Oxidases.

This study evaluates the reaction of a biomimetic heme-peroxo-copper complex, {[(DCHIm)(F8)FeIII]-(O22-)-[CuII(AN)]}+ (1), with a phenolic substrate, involving a net H-atom abstraction to cleave the bridging peroxo O-O bond that produces FeIV═O, CuII-OH, and phenoxyl radical moieties, analogous to the chemistry carried out in heme-copper oxidases (HCOs). A 3D potential energy surface generated for this reaction reveals two possible reaction pathways: one involves nearly complete proton transfer (PT) from the phenol to the peroxo ligand before the barrier; the other involves O-O homolysis, where the phenol remains H-bonding to the peroxo OCu in the transition state (TS) and transfers the H+ after the barrier. In both mechanisms, electron transfer (ET) from phenol occurs after the PT (and after the barrier); therefore, only the interaction with the H+ is involved in lowering the O-O cleavage barrier. The relative barriers depend on covalency (which governs ET from Fe), and therefore vary with DFT functional. However, as these mechanisms differ by the amount of PT at the TS, kinetic isotope experiments were conducted to determine which mechanism is active. It is found that the phenolic proton exhibits a secondary kinetic isotope effect, consistent with the calculations for the H-bonded O-O homolysis mechanism. The consequences of these findings are discussed in relation to O-O cleavage in HCOs, supporting a model in which a peroxo intermediate serves as the active H+ acceptor, and both the H+ and e- required for O-O cleavage derive from the cross-linked Tyr residue present at the active site.

A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate Complex-The Product of the Reaction of Nitrogen Monoxide (·NO(g)) with a Ferric-Superoxide Species.

Peroxynitrite (-OON═O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O2•-) with nitric oxide (·NO) and has been suggested to form an as-yet unobserved bound heme-iron-PN intermediate in the catalytic cycle of nitric oxide dioxygenase (NOD) enzymes, which facilitate a ·NO homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-FeIII complex [(PIm)FeIII(-OON═O)] (3) (PIm; a porphyrin moiety with a covalently tethered imidazole axial "base" donor ligand) has been identified and characterized by various spectroscopies (UV-vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at -80 °C by addition of ·NO(g) to the heme-superoxo species, [(PIm)FeIII(O2•-)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the PN ligand coordinated to iron via its terminal peroxidic anionic O atom with the overall geometry being in a cis-configuration. Complex 3 thermally transforms to its isomeric low-spin nitrato form [(PIm)FeIII(NO3-)] (4a). While previous (bio)chemical studies show that phenolic substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol (2,4DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H atom approaches the terminal PN O atom (farthest from the metal center and ring core), effecting O-O cleavage, giving nitrogen dioxide (·NO2) plus a ferryl compound [(PIm)FeIV═O] (7); this rebounds to give [(PIm)FeIII(NO3-)] (4a).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished.

Critical Aspects of Heme-Peroxo-Cu Complex Structure and Nature of Proton Source Dictate Metal-O(peroxo) Breakage versus Reductive O-O Cleavage Chemistry.

The 4H+/4e- reduction of O2 to water, a key fuel-cell reaction also carried out in biology by oxidase enzymes, includes the critical O-O bond reductive cleavage step. Mechanistic investigations on active-site model compounds, which are synthesized by rational design to incorporate systematic variations, can focus on and resolve answers to fundamental questions, including protonation and/or H-bonding aspects, which accompany electron transfer. Here, we describe the nature and comparative reactivity of two low-spin heme-peroxo-Cu complexes, LS-4DCHIm, [(DCHIm)F8FeIII-(O22-)-CuII(DCHIm)4]+, and LS-3DCHIm, [(DCHIm)F8FeIII-(O22-)-CuII(DCHIm)3]+ (F8 = tetrakis(2,6-difluorophenyl)-porphyrinate; DCHIm = 1,5-dicyclohexylimidazole), toward different proton (4-nitrophenol and [DMF·H+](CF3SO3-)) (DMF = dimethyl-formamide) or electron (decamethylferrocene (Fc*)) sources. Spectroscopic reactivity studies show that differences in structure and electronic properties of LS-3DCHIm and LS-4DCHIm lead to significant differences in behavior. LS-3DCHIm is resistant to reduction, is unreactive toward weakly acidic 4-NO2-phenol, and stronger acids cleave the metal-O bonds, releasing H2O2. By contrast, LS-4DCHIm forms an adduct with 4-NO2-phenol, which includes an H-bond to the peroxo O-atom distal to Fe (resonance Raman (rR) spectroscopy and DFT). With addition of Fc* (2 equiv overall required), O-O reductive cleavage occurs, giving water, Fe(III), and Cu(II) products; however, a kinetic study reveals a one-electron rate-determining process, ket = 1.6 M-1 s-1 (-90 °C). The intermediacy of a high-valent [(DCHIm)F8FeIV═O] species is thus implied, and separate experiments show that one-electron reduction-protonation of [(DCHIm)F8FeIV═O] occurs faster (ket2 = 5.0 M-1 s-1), consistent with the overall postulated mechanism. The importance of the H-bonding interaction as a prerequisite for reductive cleavage is highlighted.

A "naked" Fe(III)-(O2²â»)-Cu(II) species allows for structural and spectroscopic tuning of low-spin heme-peroxo-Cu complexes.

Here we describe a new approach for the generation of heme-peroxo-Cu compounds, using a "naked" complex synthon, [(F8)Fe(III)-(O2(2-))-Cu(II)(MeTHF)3](+) (MeTHF = 2-methyltetrahydrofuran; F8 = tetrakis(2,6-difluorophenyl)porphyrinate). Addition of varying ligands (L) for Cu allows the generation and spectroscopic characterization of a family of high- and low-spin Fe(III)-(O2(2-))-Cu(II)(L) complexes. These possess markedly varying Cu(II) coordination geometries, leading to tunable Fe-O, O-O, and Cu-O bond strengths. DFT calculations accompanied by vibrational data correlations give detailed structural insights.

Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones.

Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.

Conserved microtubule-actin interactions in cell movement and morphogenesis.

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural. These interactions comprise at least three conserved 'mechanochemical activity modules' that perform similar roles in these diverse cell functions.

The three-spin intermediate at the O-O cleavage and proton-pumping junction in heme-Cu oxidases.

Understanding the mechanistic coupling of molecular oxygen reduction and proton pumping for adenosine triphosphate synthesis during cellular respiration is the primary goal of research on heme-copper oxidases­the terminal complex in the membrane-bound electron transport chain. Cleavage of the oxygen-oxygen bond by the heme-copper oxidases forms the key intermediate PM, which initiates proton pumping. This intermediate is now experimentally defined by variable-temperature, variable-field magnetic circular dichroism spectroscopy on a previously unobserved excited state feature associated with its heme iron(IV)-oxo center. These data provide evidence that the iron(IV)-oxo in PM is magnetically coupled to both a copper(II) and a cross-linked tyrosyl radical in the active site. These results provide new insight into the oxygen-oxygen bond cleavage and proton-pumping mechanisms of heme-copper oxidases.

Multiplexed force measurements on live cells with holographic optical tweezers.

We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system's abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.

Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.

We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin-microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at approximately 1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.

L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1.

Dynamic regulation of the cell surface expression of adhesion molecules is an important mechanism for controlling neuronal growth cone motility and guidance. Clathrin-mediated vesicular internalization of L1 via the tyrosine-based endocytosis motif YRSL regulates adhesion and signaling by this Ig superfamily molecule. Here, we present evidence that tyrosine-1176 (Y1176) of the YRSL motif is phosphorylated in vivo. The nonreceptor tyrosine kinase (p60src) is implicated in L1-mediated neurite outgrowth, and we find that p60src phosphorylates Y1176 in vitro. Phosphorylation of Y1176 prevents L1 binding to AP-2, an adaptor required for clathrin-mediated internalization of L1. mAb 74-5H7 recognizes the sequence immediately NH2-terminal to the tyrosine-based motif and binds L1 only when Y1176 is dephosphorylated. 74-5H7 identifies a subset of L1 present at points of cell-cell contact and in vesicle-like structures that colocalize with an endocytosis marker. L1-L1 binding or L1 cross-linking induces a rapid increase in 74-5H7 immunoreactivity. Our data suggest a model in which homophilic binding or L1 cross-linking triggers transient dephosphorylation of the YRSL motif that makes L1 available for endocytosis. Thus, the regulation of L1 endocytosis through dephosphorylation of Y1176 is a critical regulatory point of L1-mediated adhesion and signaling.

Influence of intramolecular secondary sphere hydrogen-bonding interactions on cytochrome c oxidase inspired low-spin heme-peroxo-copper complexes.

Dioxygen reduction by heme-copper oxidases is a critical biochemical process, wherein hydrogen bonding is hypothesized to participate in the critical step involving the active-site reductive cleavage of the O-O bond. Sixteen novel synthetic heme-(µ-O2 2-)-Cu(XTMPA) complexes, whose design is inspired by the cytochrome c oxidase active site structure, were generated in an attempt to form the first intramolecular H-bonded complexes. Derivatives of the "parent" ligand (XTMPA, TMPA = (tris((2-pyridyl)methyl)amine)) possessing one or two amine pendants preferentially form an H-bond with the copper-bound O-atom of the peroxide bridge. This is evidenced by a characteristic blue shift in the ligand-to-metal charge transfer (LMCT) bands observed in UV-vis spectroscopy (consistent with lowering of the peroxo π* relative to the iron orbitals) and a weakening of the O-O bond determined by resonance Raman spectroscopy (rR), with support from Density Functional Theory (DFT) calculations. Remarkably, with the TMPA-based infrastructure (versus similar heme-peroxo-copper complexes with different copper ligands), the typically undetected Cu-O stretch for these complexes was observed via rR, affording critical insights into the nature of the O-O peroxo core for the complexes studied. While amido functionalities have been shown to have greater H-bonding capabilities than their amino counterparts, in these heme-peroxo-copper complexes amido substituents distort the local geometry such that H-bonding with the peroxo core only imparts a weak electronic effect; optimal H-bonding interactions are observed by employing two amino groups on the copper ligand. The amino-substituted systems presented in this work reveal a key orientational anisotropy in H-bonding to the peroxo core for activating the O-O bond, offering critical insights into effective O-O cleavage chemistry. These findings indirectly support computational and protein structural studies suggesting the presence of an interstitial H-bonding water molecule in the CcO active site, which is critical for the desired reactivity. The results are evaluated with appropriate controls and discussed with respect to potential O2-reduction capabilities.

Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow.

Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. We have used multimode fluorescent speckle microscopy (FSM) to directly address this problem. We report that actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, LPA mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration.

Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.

Dynamic microtubules explore the peripheral (P) growth cone domain using F actin bundles as polymerization guides. Microtubule dynamics are necessary for growth cone guidance; however, mechanisms of microtubule reorganization during growth cone turning are not well understood. Here, we address these issues by analyzing growth cone steering events in vitro, evoked by beads derivatized with the Ig superfamily cell adhesion protein apCAM. Pharmacological inhibition of microtubule assembly with low doses of taxol or vinblastine resulted in rapid clearance of microtubules from the P domain with little effect on central (C) axonal microtubules or actin-based motility. Early during target interactions, we detected F actin assembly and activated Src, but few microtubules, at apCAM bead binding sites. The majority of microtubules extended toward bead targets after F actin flow attenuation occurred. Microtubule extension during growth cone steering responses was strongly suppressed by dampening microtubule dynamics with low doses of taxol or vinblastine. These treatments also inhibited growth cone turning responses, as well as focal actin assembly and accumulation of active Src at bead binding sites. These results suggest that dynamic microtubules carry signals involved in regulating Src-dependent apCAM adhesion complexes involved in growth cone steering.