The Impact of Second Coordination Sphere Methionine-Aromatic Interactions in Copper Proteins

The interplay between the primary and secondary coordination spheresin biological metal sites plays an essential role in controlling their properties. Some ofthe clearest examples of this are from copper sites in blue and purple copper proteins.Many such proteins contain methionine (Met) in the primary coordination sphere as aweakly bound ligand to Cu. While the effects of replacing the coordinated Met areunderstood, less so is the importance of its second-sphere interactions. In thiscombined informatics and experimental study, wefirst present a bioinformaticsinvestigation of the second-sphere environments in biological Met-Cu motifs. Themost common interaction is between the Met-CH3and the pi-face of a phenylalanine(Phe) (81% of surveyed structures), tyrosine (Tyr) (11%), and tryptophan (Trp)(8%). In most cases, the Met-CH3also forms a contact with a pi-face of one of a Cu-ligating histidine-imidazole. Such interactions are widely distributed in different Cuproteins. Second, to explore the impact of the second-sphere interactions of Met, a series of artificialPseudomonas aeruginosaazurinproteins were produced where the native Phe15 was replaced with Tyr or Trp. The proteins were characterized using optical andmagnetic resonance spectroscopies, X-ray diffraction, electrochemistry, and an investigation of the time-resolved electron-transferkinetics of photosensitizer-modified proteins. The influence of the Cu-Met-Aro interaction on azurin's physical properties is subtle,and the hallmarks of the azurin blue copper site are maintained. In the Phe15Trp variant, the mutation to Phe15 induces changes inCu properties that are comparable to replacement of the weak Met ligand. The broader impacts of these widely distributed interactions are discussed

Automated summary

The interplay between the primary and secondary coordination spheres is essential for controlling the properties of biological metal sites. This study investigates the second-sphere environments in biological Met-Cu motifs and explores the impact of the second-sphere interactions on Cu properties using artificial azurin proteins. The results reveal the importance of these widely distributed interactions in controlling the physical properties of copper proteins.