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

Abstract

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 Rattus norvegicus 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, k(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.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…3). Protein preparations were found to be at least 97.5% pure and contained ϳ15% endogenously bound iron as judged by colorimetric assay using ferrozine in a 96-well format as described previously (57). Protein concentrations were determined spectrophotometrically at 280 nm using an extinction coefficient of 29,700 M Ϫ1 cm Ϫ1 estimated from a bovine serum albumin standard curve quantified by SDS-PAGE densitometry.…”

“…Phenix Refine and Readyset were used for refinement. The following residues were not included in the final model (chain A Iron Binding by PA2602 Protein-Iron binding was investigated by adding various amounts of ferrous iron sulfate to deoxygenated protein, removing the excess iron by Chelex 100, and then measuring the protein-bound iron by a ferrozine-based assay (57). The data were fitted to the equation (64,65)…”

“…Cysteine-dependent Oxygen Depletion Activity of PA2602 Protein-Activities upon the addition of cysteine to protein samples were tested by following oxygen depletion with a Clarke-type oxygen electrode (Rank Brothers) as described previously (57). Appropriate control experiments were conducted to confirm that oxygen depletion was not due to cysteine autoxidation under reaction conditions.…”

“…Mössbauer Spectroscopy-Ferrous 57 Fe stock solutions for Mössbauer spectroscopy experiments were prepared as described previously (67). Anaerobic preparations of protein (0.8 -1.5 mM) in 100 mM Tris-HCl (pH 8.0) and 50 mM NaCl were iron-saturated anaerobically by exogenously adding ferrous iron in the presence of 1 eq of dithionite, followed by treatment with analytical grade Chelex 100 sodium form, 200 -400 mesh (Bio-Rad).…”

“…Protein samples were frozen in liquid nitrogen in a glovebox (Belle Technology). 57 Fe Mössbauer spectra of frozen liquid samples in a custom Teflon sample holder (ϳ400-l volume) were recorded on a Mössbauer spectrometer from Science Engineering & Education Co. (Edina, MN) equipped with a closed cycle refrigerator system from Janis Research Co. and Sumitomo Heavy Industries Ltd. and a temperature controller from Lakeshore Cryotronics, Inc. Data were collected in constant acceleration mode in transmission geometry with an applied field of 47 milliteslas parallel to the ␥-rays.…”