Journal
BIOCHEMISTRY
Volume 58, Issue 17, Pages 2218-2227Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.9b00006
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Funding
- National Science Foundation [CHE-1808637]
- National Institutes of Health [P41GM103393, GM107529]
- Lutcher Brown Distinguished Chair Endowment fund
- Advanced Photon Source Section 19, Structural Biology Center user program, Argonne National Laboratory [GUP-48198]
- Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory [5B14]
- DOE Office of Science [DE-AC02-06CH11357]
- U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
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Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, 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 cross-linking 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 uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-L-tyrosine (F-2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide ((NO)-N-center dot), an oxygen surrogate, similarly binds to uncross-linked F-2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F-2-Tyr157 CDO and mature wild-type CDO in complex with both L-cysteine and (NO)-N-center dot. 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 structure-based understanding is further supported by a computational study with models built on the uncross-linked 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 cross-link formation is proposed.
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