The function and characteristics of tyrosyl radical cofactors.

Amino-acid radicals are involved in the catalytic cycles of a number of enzymes. The main focus of this mini-review is to discuss the function and properties of tyrosyl radical cofactors. We start by briefly summarizing the experimental studies that led to the detection and identification of the two redox-active tyrosines, denoted Y(Z) and Y(D), found in the water-oxidizing photosystem II (PSII) enzyme. More recent work that shows that the histidine-cross-linked tyrosine located in the active site of cytochrome c oxidase forms a radical during the catalytic oxygen-oxygen bond-cleavage process is also described. Advanced spectroscopic and structural studies have been performed to investigate the spin-density distribution, the protonation state and the hydrogen bonding of redox-active tyrosines. These studies have shown that the radical spin-density distribution is highly insensitive to the environment and that it is typical of a deprotonated species. In contrast, the hydrogen bonding and the nature of the proton acceptor or network of acceptors vary substantially in different systems. This is important for the function of the tyrosyl radical, as will be emphasized in a detailed discussion on the proposed function of Y(Z) as a proton coupled electron-transfer cofactor in photosynthetic water oxidation. Amino-acid radical enzymes are typically large complexes containing multiple subunits, chromophores and redox cofactors. The structural and mechanistic complexity of these systems has hampered the detailed characterization of their radical cofactors. In the final section of this mini-review, we will describe a project aimed at investigating how the protein controls the thermodynamic and kinetic redox properties of aromatic residues by using de novo protein design. Two model proteins of different size have been constructed. The smaller protein is a 67-residue three-helix bundle containing either a single buried tryptophan or tyrosine residue. The high-resolution NMR structure of the tryptophan-containing protein, denoted alpha(3)W, shows that the aromatic side chain is involved in a pi-cation interaction with a nearby lysine. The effects of this interaction on the tryptophan reduction potential were investigated by electrochemical and quantum mechanical methods. The calculations predict that the pi-cation interaction increases the potential, which is consistent with the electrochemical characterization of alpha(3)W. A larger 117-residue four-helix bundle, alpha(4)W, has more recently been constructed to complement the work on the three-helix-bundles and expand the family of model radical proteins.