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

Abstract

The apo-protein of Pseudomonas aeruginosa 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 dz2 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.

Density Functional Calculations for Prediction of 57Fe Mössbauer Isomer Shifts and Quadrupole Splittings in β-Diketiminate Complexes

“…8 These showed us that the existing correlations have few examples of multimetallic complexes, redox-active ligands, low-valent, and low-coordinate iron complexes. Additionally, the established correlations use continuum solvation models with the polarity of water 9b or methanol, 9d whereas the iron complexes of interest to our group are often studied experimentally in less polar solvents, such as toluene.…”

Electrochemical, Spectroscopic, and Density Functional Theory Characterization of Redox Activity in Nickel-Substituted Azurin: A Model for Acetyl-CoA Synthase

“…Previous reports of iron substitution in azurin suggested that redox activity could only be achieved by altering the ligands in the binding site or using nuclear irradiation techniques such as cryoreduction. 27,60,61 Importantly, the Ni II/I couple remains physiologically accessible. Metalloenzymes containing low-valent nickel active sites have comparable reduction potentials, closely matched to the chemistry performed by those enzymes; the reduction potential of the proximal nickel center in ACS is observed at −540 mV.…”

An internal electron reservoir enhances catalytic CO2 reduction by a semisynthetic enzyme

“…Az has been used in many protein engineering studies and is robust towards mutation, serving as a tunable platform that imposes a well-defined secondary coordination sphere, which can modulate catalytic efficiency and selectivity. 20,21 …”