THE DE-NOVO DESIGN OF A RUBREDOXIN-LIKE FE SITE

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally c oordinated by four cysteine residues, [Fe(S-Cys)(4)](-1,-2). The desig n criteria for the target site included taking backbone movements into account, tetrahedral metal-binding and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(II) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism an d nuclear magnetic resonance measurements reveal that the metal-free a nd Cd(II)-bound forms of the variant are folded correctly and are stab le. The Fe(III) complex of the metal-binding mutant reproduces the opt ical and the electron paramagnetic resonance spectra of oxidized rubre doxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.