Kβ Valence to Core X-ray Emission Studies of Cu(I) Binding Proteins with Mixed Methionine – Histidine Coordination. Relevance to the Reactivity of the M- and H-sites of Peptidylglycine Monooxygenase

Abstract

Biological systems use copper as a redox center in many metalloproteins, where the role of the metal is to cycle between its +1 and +2 oxidation states. This chemistry requires the redox potential to be in a range that can stabilize both Cu(I) and Cu(II) states, and often involves protein-derived ligand sets involving mixed histidine-methionine coordination that balance the preferences of both oxidation states. Transport proteins, on the other hand, utilize copper in the Cu(I) state, and often contain sites comprised predominately of the cuprophilic residue methionine. The electronic factors that allow enzymes and transporters to balance their redox requirements are complex, and are often elusive due to the dearth of spectroscopic probes of the Cu(I) state. Here we present the novel application of X-ray emission spectroscopy to copper proteins via a study of a series of mixed His - Met copper sites where the ligand set varies in a systematic way between the His3 and Met3 limits. The sites are derived from the wild-type peptidylglycine monooxygenase (PHM), two single-site variants which replicate each of its two copper sites (CuM-site and CuH-site), and the transporters CusF and CusB. Clear differences are observed in the Kβ2,5 region at the Met3 and His3 limits. CusB (Met3) has a distinct peak at 8978.4 eV with a broad shoulder at 8975.6 eV, whereas CuH (His3) has two well-resolved features: a more intense feature at 8974.8 eV and a second at 8977.2 eV. The mixed coordination sphere CusF (Met2His) and the PHM CuM variant (Met1His2) have very similar spectra consisting of two features at 8975.2 eV and 8977.8 eV Analysis of DFT calculated spectra indicate that the intensity of the higher energy peak near 8978 eV is mediated by mixing of ligand-based orbitals into the Cu d10 manifold, with S from Met providing more intensity by facilitating increased Cu p-d mixing. Furthermore, reaction of WT PHM with CO (an oxygen analogue) produced the M-site CO complex, which showed a unique XES spectrum that could be computationally reproduced by including interactions between Cu(I) and the CO ligand. The study suggests that the valence-to-core (VtC) region can serve as a probe of not only ligand speciation, but also offer insight into the coordination geometry, in a fashion similar to XAS pre-edges, and may be sufficiently sensitive to the coordination of exogenous ligands to be useful in the study of reaction mechanisms.

Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase

“…This is an attractive idea, since the Met ligand is essential to catalysis 21, 35, 36 . A recent X-ray emission study of PHM 37 has emphasized an important role for d-p mixing between the filled d 10 manifold and both S-Met and N-His ligands, with metal-d:ligand-S overlap predominating. Therefore differences in Cu-S(Met) orientation could have significant effects on back-bonding interactions.…”

Structural Elucidation, Aggregation, and Dynamic Behaviour of N,N,N,N‐Copper(I) Schiff Base Complexes in Solid and in Solution: A Combined NMR, X‐ray Spectroscopic and Crystallographic Investigation

“…Motivated by the consistent quantitative agreement between experimental and calculated VtC spectra demonstrated in previous studies, [72][73][74][75][76][77] calculations on DFT optimized structures were pursued. Based on the evidence from the Cu K-edge XAS that 1 and 5 are tetracoordinated, models of the Δ atropisomer of the cations of 1 and 5 were built.…”

Phosphorus Kβ X-ray emission spectroscopy detects non-covalent interactions of phosphate biomolecules in situ

“…Contributions from the filled 3d orbitals may also be observed, provided there is sufficient metal p character in these MOs. [78][79][80] Third-row main group elements differ from first-row transition metals in that their valence orbitals, those most capable of mixing with ligand orbitals, are 3p rather than 3d. Thus, phosphorus Kβ mainline transitions are not approximately atomic, but rather reflect the valence bonding environment analogous to transition metal VtC transitions while being more strongly dipole-allowed.…”