JBIC Journal of Biological Inorganic Chemistry

, Volume 14, Issue 6, pp 913–921 | Cite as

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

  • Torsten Kleffmann
  • Seino A. K. Jongkees
  • Graham Fairweather
  • Sigurd M. Wilbanks
  • Guy N. L. Jameson
Original Paper

Abstract

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes.

Keywords

Cysteine dioxygenase Oxygen Iron Cystine Non-heme monoiron 

Abbreviations

CDO

Cysteine dioxygenase

CID

Collision-induced dissociation

ISD

In-source decay

LC

Liquid chromatography

MALDI

Matrix-assisted laser desorption/ionization

MS

Mass spectrometry

PAGE

Polyacrylamide gel electrophoresis

SDS

Sodium dodecyl sulfate

TFA

Trifluoroacetic acid

Supplementary material

775_2009_504_MOESM1_ESM.pdf (4.4 mb)
Supplementary material 1 (PDF 4526 kb)

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Copyright information

© SBIC 2009

Authors and Affiliations

  • Torsten Kleffmann
    • 1
    • 2
  • Seino A. K. Jongkees
    • 2
    • 3
  • Graham Fairweather
    • 3
  • Sigurd M. Wilbanks
    • 2
  • Guy N. L. Jameson
    • 3
  1. 1.Department of Biochemistry, Centre for Protein ResearchUniversity of OtagoDunedinNew Zealand
  2. 2.Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  3. 3.Department of ChemistryUniversity of OtagoDunedinNew Zealand

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