K ss Detected High-Resolution XANES of FeII and FeIII Models of the 2-His-1-Carboxylate Motif: Analysis of the Carboxylate Binding Mode

Abstract

Proteins sharing the same 2-His-1-carboxylate structural motif have little amino acid sequence similarity and are able to perform many different reactions. Many factors have been cited to explain their different specificity and turnover rates, like protein environment, coordinated ligand geometry, electronic structure of the active site, etc. In this paper, we present a combined approach applying high-resolution XANES spectroscopy and theory simulations to different model complexes that mimic the binding modes of the amino acids to the metal site. Experiments were performed on three compounds showing three metal sites: ferrous hexacoordinate, ferric pentacoordinate and ferrous pentacoordinate. The first two compounds bear an N,N,O-tridentate 3,3-bis(1-alkylimidazol-2-yl)propionate ligand that features a monodentate carboxylate group. These complexes mimic the activity of extradiol dioxygenases but also exhibit intradiol cleavage activity. The third compound features a bidentate terphenylcarboxylate ligand and a sterically hindered bidentate N,N-donor, thus providing a good structural mimic of the ternary enzyme-tetrahydrobiopterin-substrate complex in pterin-dependent phenylalanine hydroxylase, which also contains a bidentate carboxylate. Modeling of high-resolution XANES on well-defined model complexes of different geometry can aid in protein structure elucidation. XANES gives the oxidation state and coordination number of the metal in the non-crystallized protein at natural pH. The accuracy of the results is limited by the core-hole and experimental broadenings. We found that high-resolution XANES experiments give increased resolution at the pre-edges, but limited improvement at the main edge. These high-resolution pre-edges can be accurately simulated by using crystal field multiplet theory (CFM). We show that by combining modelling and XANES simulations with FEFF8, detailed structural and chemical information can be obtained. We found that a short O2metal distance for the carboxylate oxygen atom not bound to the metal causes a higher white line in FeII, which is similar to the results obtained for the pterin-dependent hydroxylase, tyrosine hydroxylase (TYH). Full-potential FDMNES simulations for each sample confirm the accuracy of the main results with muffin-tin approximation (FEFF8).