Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens.

Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active PlAA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu g-tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of g-tensors, giving confidence in our evaluation of PlAA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of PlAA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in PlAA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.

Unstability of cinnabarinic acid, an endogenous metabolite of tryptophan, under situations mimicking physiological conditions.

The kynurenine pathway of l-tryptophan metabolism produces several compounds of high physiological importance in the central nervous system and the immune response. Among them, cinnabarinic acid (CA) which results from the condensation of two molecules of 3-hydroxy-anthranilic acid has been identified as an activator of the metabotropic glutamate receptor (mGluR4) and the aryl hydrocarbon receptor (AhR). However, very few information was available about its stability under physiological conditions. This article shows that CA is unstable even under very soft conditions mimicking physiological conditions. Incubations in phosphate buffer pH 7.4 lead to several products coming from various reactions such as addition of H2O on its quinone imine function, decarboxylation, and deamination. Moreover, CA rapidly reacts with glutathione (GSH), leading to adducts that result from the Michael type addition of this physiological nucleophile on the quinone imine function of CA. These preliminary results indicate that the great reactivity of CA and the nature of its various products should be considered when studying its activity towards any biological target.

Comparison of Density Functional and Correlated Wave Function Methods for the Prediction of Cu(II) Hyperfine Coupling Constants.

The reliable prediction of Cu(II) hyperfine coupling constants remains a challenge for quantum chemistry. Until recently only density functional theory (DFT) could target this property for systems of realistic size. However, wave function based methods become increasingly applicable. In the present work, we define a large set of Cu(II) complexes with experimentally known hyperfine coupling constants and use it to investigate the performance of modern quantum chemical methods for the prediction of this challenging spectroscopic parameter. DFT methods are evaluated against orbital-optimized second-order Møller-Plesset (OO-MP2) theory and coupled cluster calculations including singles and doubles excitations, driven by the domain-based local pair natural orbital approach (DLPNO-CCSD). Special attention is paid to the definition of a basis set that converges adequately toward the basis set limit for the given property for all methods considered in this study, and a specifically optimized basis set is proposed for this purpose. The results suggest that wave function based methods can supplant but do not outcompete DFT for the calculation of Cu(II) hyperfine coupling constants. Mainstream hybrid functionals such as B3PW91 remain on average the best choice.