A Tale of Two Chromophores: Investigating Two Distinct Isoforms of Cysteine Dioxygenase

Abstract

Cysteine dioxygenase (CDO) is a mononuclear iron-containing enzyme that converts L-cysteine to L-cysteine sulfinic acid. The active site iron of CDO is coordinated by 3-His residues, and within 3.3Å of the iron center exists a unique thioether Cys-Tyr crosslink [1]. Wild-type CDO exists in a heterogeneous mixture of crosslinked and noncrosslinked species. Recent studies suggest the Cys-Tyr crosslink enhances catalytic activity, and plays a role in stabilizing the iron center through second-sphere interactions [2]. Two isoforms of CDO with distinct colors were identified in purifications of wild-type CDO. The appearance of the two CDO isoforms was observed in eluted fractions from an anion exchange column. Separate elution patterns for the two chromophoric isoforms suggest the species exist in different ionization states. The two isoforms had a similar amount of iron bound, and there was no significant difference in the steady-state kinetic parameters between the isoforms when monitoring oxygen consumption. The EPR spectra showed a high spin ferric signal for both isoforms, but there was a noticeable 4-fold decrease in the relative peak intensity for one chromophoric species. The results from the EPR studies suggest that the metal center of the two chromophoric species have different oxidations states due to altered reduction potentials generated by second-sphere charge-transfer interactions. Identifying the amino acid residues that participate in the charge transfer interactions will provide valuable insight regarding the diverging reduction potentials of the two chromophoric species.