Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket

Abstract

The apoprotein of <i>Pseudomonas aeruginosa</i> azurin binds iron­(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well as X-ray crystallography and quantum-chemical computations. Despite potential competition by water and other coordinating residues, iron­(II) binds tightly to the low-coordinate site. The iron­(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Spectroscopically calibrated quantum-chemical computations show that the complex has high-spin iron­(II) in a pseudotetrahedral coordination environment, which features interactions with side chains of two histidines and a cysteine as well as the CO of Gly45. In the ⁵A₁ ground state, the <i>d</i>_<i>z</i>² orbital is doubly occupied. Mutation of Met121 to Ala leaves the metal site in a similar environment but creates a pocket for reversible binding of small anions to the iron­(II) center. Specifically, azide forms a high-spin iron­(II) complex and cyanide forms a low-spin iron­(II) complex