Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine

Abstract

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, a posttranslationally generated Cys-Tyr cofactor is present, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the crosslinking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of L-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncrosslinked human CDO due to site-specific incorporation 3,5-difluoro-L-tyrosine (F 2 -Tyr) at the crosslinking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncrosslinked F 2 -Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncrosslinked F 2 -Tyr157 CDO and mature wild-type CDO in complex with both L-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well aligned to be oxidized should the normal oxidation reaction uncouple. This structural based understanding is further supported by a computational study with models built on the uncrosslinked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the crosslink formation is proposed.

Chemical modifications of proteins and their applications in metalloenzyme studies

“…During the crosslink biogenesis, the sulfur atom of Cys93 might rotate toward the iron center, which is supported by a recently reported structure of the uncross-linked F 2 -TyrCDO• L-Cys•NO ternary complex ( Fig. 10 e) [ 110 ]. This structure revealed an interaction between NO and one conformer of Cys93, suggesting that cysteine oxidation may occur prior to the crosslink formation.…”

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

“…The increased flexibility of ADO may arise from the requirement of ADO to handle more diverse protein substrates, while the PCOs are more specific to ERF-VIIs, with each PCO isoform having a particular substrate preference (41). In contrast, ADO is less similar to other thiol dioxygenases, with rmsd values of 2.38 Å over 152 Cα and 2.53 Å over 154 Cα for CDO and MDO, respectively (9,27). This is anticipated since both ADO and PCO oxidize protein substrates, while the corresponding structural features are superfluous for other small-molecule thiol dioxygenases.…”

“…It exhibits the β-barrel fold typical of the cupin superfamily with a Ni-substituted metal center and a cross-linked Cys-Tyr cofactor ( 5 ), which is identical in coordination to the Fe-containing form determined independently ( 6 ). Later, more CDO structures became available, such as structures of human, rat, and bacterial versions, proteins complexed with the substrate, gas molecules, as well as the structures with halogenated cofactors and without the cross-linked cofactors ( 6 , 23 , 25 , 26 , 27 , 28 , 29 ). The structures of PCO isoforms from Arabidopsis thaliana were reported very recently, including PCO2, PCO4, and PCO5, with a ligand-free Fe or Ni center ( 30 , 31 ).…”

Emerging roles for thiol dioxygenases as oxygen sensors

“…Cys 93 variants of CDO, as well as the Tyr157Phe variant, have been shown to be functional but with decreased activity [22][23][24]. Cross-link formation is proposed to be initiated by O 2 activation at the active site resulting in oxidation of Cys 93 instead of L-Cys (substrate) to generate a thiyl radical, which subsequently oxidises Tyr 157 to form the crosslink [25]. Although the mechanism by which the Cys 93 -Tyr 157 crosslink enhances enzyme kinetics is not definitive, current evidence indicates that it is through stabilisation of the Tyr 157 -OH group, which forms part of a hydrogen bonding network across the Ser 153 -His 155 -Tyr 157 motif [13,14,18], such that it is at a favourable distance and position to Accepted Article interact with the substrate as well as O 2 , and may play a role in catalysis of substrate oxidation [26,27].…”