A Strongly Bound High-Spin Iron(II) Coordinates Cysteine and Homocysteine in Cysteine Dioxygenase

The first experimental evidence of a tight binding iron­(II)–CDO complex is presented. These data enabled the relationship between iron bound and activity to be explicitly proven. Cysteine dioxygenase (CDO) from <i>Rattus norvegicus</i> has been expressed and purified with ∼0.17 Fe/polypeptide chain. Following addition of exogenous iron, iron determination using the ferrozine assay supported a very tight stoichiometric binding of iron with an extremely slow rate of dissociation, <i>k</i>_off ∼ 1.7 × 10^–6 s^–1. Dioxygenase activity was directly proportional to the concentration of iron. A rate of cysteine binding to iron­(III)–CDO was also measured. Mössbauer spectra show that in its resting state CDO binds the iron as high-spin iron­(II). This iron­(II) active site binds cysteine with a dissociation constant of ∼10 mM but is also able to bind homocysteine, which has previously been shown to inhibit the enzyme.