X-ray absorption and emission spectroscopies as probes of copper sites in metalloenzymes and model complexes
Keywords: Chemistry.

Cu enzymes play important roles in a wide range of biological processes including O2 activation. Cu active sites in these enzymes have unique geometric and electronic structures that give rise to characteristic spectral features. Thus, the investigation of these spectral features provides insight into the structure--function correlation in Cu enzymes. The focus of this thesis is to utilize X-ray absorption and emission spectroscopies to probe Cu sites in metalloenzymes and model complexes. Chapter 1: This chapter provides the background of mononuclear Cu enzymes and Cu corroles, and the introduction to the methodologies of X-ray absorption and emission spectroscopies. Chapter 2: Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kβ X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kβ XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double ionization (DI) states are identified using varying incident X-ray photon energies and a reasonable method to correct the data to remove DI contributions is presented. Kβ XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kβ2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kβ2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d--p mixing. A Kβ XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kβ2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2−y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kβ XES for probing the Cu(I) FMO associated with small molecule activation in metalloproteins. Chapter 3: The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-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 O2 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 O2 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. Chapter 4: The formylglycine-generating enzyme (FGE) catalyzes the O2-dependent conversion of Cys to formylglycine. The Cu(I) active site of the FGE is coordinated by two Cys residues in a linear geometry. With substrate binding, the Cu(I) active site becomes a trigonal planar geometry with three Cys ligands, which turns on O2 activation. The Cu(I) active site structure in the FGE is unique because the similar bis-cysteine coordination is typically observed in Cu trafficking and sensing enzymes but not in Cu-dependent oxidases/oxygenases. Here, we apply Kβ X-ray emission spectroscopy to study the Cu(I)-FGE and the substrate bound Cu(I)-FGE active sites to probe its frontier molecular orbital(s) (FMO(s)) for O2 binding and activation. Small spectral differences are observed between Cu(I)-FGE and substrate bound Cu(I)-FGE that are correlated to the site structure change due to the substrate binding to the Cu(I) using DFT calculations. Based on these Kβ XES spectra and calculations, the Cu 3dx2−y2 vs 3dz2 nature of the FMO for O2 binding and activation, equatorially vs axially is discussed. Chapter 5: 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. Appendix 1: A macrocyclic ligand (L4−) 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)] (1) or (Me4N)[LCu2(μ-OH)] (2), depending on conditions. Complex 2 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. Appendix 2: 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. Here, 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. Appendix 3: 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