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

Biochemistry. 2019 Apr 30;58(17):2218-2227. doi: 10.1021/acs.biochem.9b00006. Epub 2019 Apr 19.

Abstract

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 (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalytic Domain
  • Cross-Linking Reagents / chemistry
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Cysteine Dioxygenase / chemistry*
  • Cysteine Dioxygenase / genetics
  • Cysteine Dioxygenase / metabolism
  • Dipeptides / chemistry*
  • Dipeptides / metabolism
  • Electron Spin Resonance Spectroscopy
  • Humans
  • Models, Molecular
  • Nitric Oxide / chemistry
  • Nitric Oxide / metabolism
  • Oxidation-Reduction
  • Oxygen / chemistry
  • Oxygen / metabolism
  • Protein Binding
  • Protein Conformation*
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine / genetics
  • Tyrosine / metabolism

Substances

  • Cross-Linking Reagents
  • Dipeptides
  • cysteinyltyrosine
  • Nitric Oxide
  • 3,5-difluorotyrosine
  • Tyrosine
  • Cysteine Dioxygenase
  • Cysteine
  • Oxygen