Hyperfine-Shifted ¹³C Resonance Assignments in an Iron−Sulfur Protein with Quantum Chemical Verification: Aliphatic C−H···S 3-Center−4-Electron Interactions

Abstract

Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar N−H or O−H moieties and O atoms or aromatic π electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker C−H···O hydrogen bonds, including ones involving sp ³-hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker C−H···S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp ³-hybridized) C−H···S 3-center−4-electron interactions in the protein Clostridium pasteurianum rubredoxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: C−H···S−Fe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule.