Proximal Methionine Amino Acid Residue Affects the Properties of Redox-Active Tryptophan in an Artificial Model Protein.

Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in disease states (e.g., oxidative-stress-associated disorders). Tryptophan (Trp) is one such redox-active amino acid residue, and it has long been known to serve a functional role in proteins. Broadly speaking, there is still much to learn about the local features that make some Trp redox active and others inactive. Herein, we describe a new protein model system where we investigate how a methionine (Met) residue proximal to a redox-active Trp affects its reactivity and spectroscopy. We use an artificial variant of azurin from Pseudomonas aeruginosa to produce these models. We employ a series of UV-visible spectroscopy, electrochemistry, electron paramagnetic resonance, and density functional theory experiments to demonstrate the effect that placing Met near Trp radicals has in the context of redox proteins. The introduction of Met proximal to Trp lowers its reduction potential by ca. 30 mV and causes clear shifts in the optical spectra of the corresponding radicals. While the effect may be small, it is significant enough to be a way for natural systems to tune Trp reactivity.

On the roles of methionine and the importance of its microenvironments in redox metalloproteins.

The amino acid residue methionine (Met) is commonly thought of as a ligand in redox metalloproteins, for example in cytochromes c and in blue copper proteins. However, the roles of Met can go beyond a simple ligand. The thioether functional group of Met allows it to be considered as a hydrophobic residue as well as one that is capable of weak dipolar interactions. In addition, the lone pairs on sulphur allow Met to interact with other groups, inluding the aforementioned metal ions. Because of its properties, Met can play diverse roles in metal coordination, fine tuning of redox reactions, or supporting protein structures. These roles are strongly influenced by the nature of the surrounding medium. Herein, we describe several common interactions between Met and surrounding aromatic amino acids and how they affect the physical properties of both copper and iron metalloproteins. While the importance of interactions between Met and other groups is established in biological systems, less is known about their roles in redox metalloproteins and our view is that this is an area that is ready for greater attention.

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

The interplay between the primary and secondary coordination spheres in biological metal sites plays an essential role in controlling their properties. Some of the 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 a weakly bound ligand to Cu. While the effects of replacing the coordinated Met are understood, less so is the importance of its second-sphere interactions. In this combined informatics and experimental study, we first present a bioinformatics investigation of the second-sphere environments in biological Met-Cu motifs. The most common interaction is between the Met-CH3 and the π-face of a phenylalanine (Phe) (81% of surveyed structures), tyrosine (Tyr) (11%), and tryptophan (Trp) (8%). In most cases, the Met-CH3 also forms a contact with a π-face of one of a Cu-ligating histidine-imidazole. Such interactions are widely distributed in different Cu proteins. Second, to explore the impact of the second-sphere interactions of Met, a series of artificial Pseudomonas aeruginosa azurin proteins were produced where the native Phe15 was replaced with Tyr or Trp. The proteins were characterized using optical and magnetic resonance spectroscopies, X-ray diffraction, electrochemistry, and an investigation of the time-resolved electron-transfer kinetics 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 in Cu properties that are comparable to replacement of the weak Met ligand. The broader impacts of these widely distributed interactions are discussed.