|dc.description.abstract||Cysteine dioxygenase (CDO) is a mononuclear ferrous enzyme located at a branch-point in cysteine metabolism and catalyzes the oxidation of L-cysteine using both atoms of dioxygen. Although several studies have attempted to characterize the enzyme kinetically, efforts have been thwarted by the apparent inactivation of the purified protein. Publication of the three-dimensional structure of several mammalian CDO enzymes revealed two unusual structural attributes that had previously not been identified in other mononuclear ferrous-dependent enzymes. The metal center of CDO is bound by a facial triad composed entirely of histidine residues in contrast to the previously ubiquitous 2-His/1-carboxylate facial triad found in other ferrous-dependent enzymes. Interestingly, a covalent linkage between Cys93 and Tyr157 was found near the metal center in the active site. The purpose of these studies is to probe the cause of inactivation of purified CDO and to determine how the 3-His facial triad and Cys-Tyr crosslink affect the catalytic properties of the enzyme.
Other ferrous-dependent enzymes stabilize the active oxidation state of their metal centers using a variety of strategies. To determine if inactivation of CDO occurring during purification is the result of an inability of the enzyme to protect its metal center from oxidation, EPR spectra and activity measurements were made of CDO expressed in cell lysate and following purification. The spectrum of CDO expressed in cell lysate was consistent with Fe(II) and the lysate displayed considerable catalytic activity for cysteine oxidation. Following purification, the EPR spectrum of CDO indicated that greater than 99% of the iron center had been oxidized to the inactive Fe(III) form. Consistent with the oxidation, purified CDO displayed no activity beyond the background oxidation of L-cysteine. Activity was recoverable by the addition of L-ascorbate to assays and this corresponded to reduction of the metal center as observed by EPR. Necessity of including an external reductant for the metal center was extended to substrate binding studies using nitric oxide as a spectrally active analog of dioxygen. Purified CDO in the resting state was capable of binding L-cysteine but incapable of binding nitric oxide; however, when preincubated with an external reductant, the metal center was reactive to both substrates.
Typical purifications of CDO yield a mixture of crosslinked and non-crosslinked isoforms of the enzyme. Previous studies have shown that a single isoform is responsible for all observable activity. To determine which isoform represented the physiologically active enzyme, a two-fold approach was taken. Site-directed mutagenesis was used to replace Cys93 and Tyr157 with serine and phenylalanine, respectively, in order to study CDO in the absence of the thioether linkage. Both variant CDO enzymes were capable of binding substrates and retained catalytic efficiencies comparable to the wild-type enzyme, suggesting that the crosslink was not necessary for catalysis. To study CDO containing the thioether linkage, a method was developed to isolate the enzyme in the homogenously crosslinked isoform. The resulting homogenously crosslinked enzyme was capable of binding both substrates but was catalytically inactive. This confirms the previous observation that only one isoform of CDO is catalytically active and demonstrates that the active isoform is non-crosslinked CDO. EPR spectroscopy was used to show that the metal center of homogenously crosslinked CDO has an altered metal coordination environment compared to wild-type, C93S, or Y157F CDO proteins, and this results in release of the metal center from the active site. The activity of CDO in vivo has been shown to be tightly regulated. The combined sensitivity of CDO to inactivation by oxidation of the metal center and crosslink formation may be linked physiologically and represent a redox sensitive regulation of CDO activity.||en