Metalloprotein entatic control of ligand-metal bonds quantified by ultrafast x-ray spectroscopy

The multifunctional protein cytochrome c (cyt c) plays key roles in electron transport and apoptosis, switching function by modulating bonding between a heme iron and the sulfur in a methionine residue. This Fe-S(Met) bond is too weak to persist in the absence of protein constraints. We ruptured the bond in ferrous cyt c using an optical laser pulse and monitored the bond reformation within the protein active site using ultrafast x-ray pulses from an x-ray free-electron laser, determining that the Fe-S(Met) bond enthalpy is ~4 kcal/mol stronger than in the absence of protein constraints. The 4 kcal/mol is comparable with calculations of stabilization effects in other systems, demonstrating how biological systems use an entatic state for modest yet accessible energetics to modulate chemical function

X-ray Absorption Spectroscopy as a Probe of Ligand Noninnocence in Metallocorroles: The Case of Copper Corroles

The question of ligand noninnocence in Cu corroles has long been a topic of discussion. Presented herein is a Cu K-edge X-ray absorption spectroscopy (XAS) study, which provides a direct probe of the metal oxidation state, of three Cu corroles, Cu[TPC], Cu[Br8TPC], and Cu[(CF3)8TPC] (TPC = meso-triphenylcorrole), and the analogous Cu(II) porphyrins, Cu[TPP], Cu[Br8TPP], and Cu[(CF3)8TPP] (TPP = meso-tetraphenylporphyrin). The Cu K rising-edges of the Cu corroles were found to be about 0−1 eV upshifted relative to the analogous porphyrins, which is substantially lower than the 1−2 eV shifts typically exhibited by authentic Cu(II)/Cu(III) model complex pairs. In an unusual twist, the Cu K pre-edge regions of both the Cu corroles and the Cu porphyrins exhibit two peaks split by 0.8− 1.3 eV. Based on time-dependent density functional theory calculations, the lower- and higher-energy peaks were assigned to a Cu 1s → 3dx2−y2 transition and a Cu 1s → corrole/porphyrin π* transition, respectively. From the Cu(II) porphyrins to the corresponding Cu corroles, the energy of the Cu 1s → 3dx2−y2 transition peak was found to upshift by 0.6−0.8 eV. This shift is approximately half that observed between Cu(II) to Cu(III) states for well-defined complexes. The Cu K-edge XAS spectra thus show that although the metal sites in the Cu corroles are more oxidized relative to those in their Cu(II) porphyrin analogues, they are not oxidized to the Cu(III) level, consistent with the notion of a noninnocent corrole. The relative importance of σ-donation versus corrole π-radical character is discussed

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-Fe^III complex [(P^Im)­Fe^III(⁻OONO)] (<b>3</b>) (P^Im; 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, [(P^Im)­Fe^III(O₂^•–)] (<b>2</b>). DFT calculations confirm that <b>3</b> 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 <i>cis</i>-configuration. Complex <b>3</b> thermally transforms to its isomeric low-spin nitrato form [(P^Im)­Fe^III(NO₃^–)] (<b>4a</b>). 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-<i>tert</i>-butylphenol (^2,4DTBP) to complex <b>3</b> does not lead to nitrated phenol; the nitrate complex <b>4a</b> 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 [(P^Im)­Fe^IVO] (<b>7</b>); this rebounds to give [(P^Im)­Fe^III(NO₃^–)] (<b>4a</b>).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished

Hydroxo-Bridged Dicopper(II,III) and -(III,III) Complexes: Models for Putative Intermediates in Oxidation Catalysis

A macrocyclic ligand (L^4–) comprising two pyridine­(dicarboxamide) donors was used to target reactive copper species relevant to proposed intermediates in catalytic hydrocarbon oxidations by particulate methane monooxygenase and heterogeneous zeolite systems. Treatment of LH₄ with base and Cu­(OAc)₂·H₂O yielded (Me₄N)₂[L₂Cu₄(μ₄-O)] (<b>1</b>) or (Me₄N)­[LCu₂(μ-OH)] (<b>2</b>), depending on conditions. Complex <b>2</b> was found to undergo two reversible 1-electron oxidations via cyclic voltammetry and low-temperature chemical reactions. On the basis of spectroscopy and theory, the oxidation products were identified as novel hydroxo-bridged mixed-valent Cu­(II)­Cu­(III) and symmetric Cu­(III)₂ species, respectively, that provide the first precedence for such moieties as oxidation catalysis intermediates

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O 2 activation

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C -formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation

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-Fe^III complex [(P^Im)­Fe^III(⁻OONO)] (<b>3</b>) (P^Im; 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, [(P^Im)­Fe^III(O₂^•–)] (<b>2</b>). DFT calculations confirm that <b>3</b> 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 <i>cis</i>-configuration. Complex <b>3</b> thermally transforms to its isomeric low-spin nitrato form [(P^Im)­Fe^III(NO₃^–)] (<b>4a</b>). 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-<i>tert</i>-butylphenol (^2,4DTBP) to complex <b>3</b> does not lead to nitrated phenol; the nitrate complex <b>4a</b> 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 [(P^Im)­Fe^IVO] (<b>7</b>); this rebounds to give [(P^Im)­Fe^III(NO₃^–)] (<b>4a</b>).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished