title: Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron.
abstract: Cysteine dioxygenase (CDO) utilizes a 3-His facial triad for coordination of its metal center. Recombinant CDO present in cellular lysate exists primarily in the ferrous form and exhibits significant catalytic activity. Removal of CDO from the reducing cellular environment during purification results in the loss of bound iron and oxidation of greater than 99% of the remaining metal centers. The as-isolated recombinant enzyme has comparable activity as the background level of L-cysteine oxidation confirming that CDO is inactive under the aerobic conditions required for catalysis. Including exogenous ferrous iron in assays resulted in non-enzymatic product formation; however, addition of an external reductant in assays of the purified protein resulted in the recovery of CDO activity. EPR spectroscopy of CDO in the presence of a reductant confirms that the recovered activity is consistent with reduction of iron to the ferrous form. The as-isolated enzyme in the presence of L-cysteine was nearly unreactive with the dioxygen analog, but had increased affinity when pre-incubated with an external reductant. These studies shed light on the discrepancies among reported kinetic parameters for CDO and also juxtapose the stability of the 3-His and 2-His/1-carboxylate ferrous enzymes in the presence of dioxygen.
authors.affiliation: Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Imsand
authors.forename: Catherine W
authors.initials: CW
authors.lastname: Njeri
authors.forename: Holly R
authors.initials: HR
authors.lastname: Ellis
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D050558:Cysteine Dioxygenase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D050558:Cysteine Dioxygenase