Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase.
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.
Department of Biochemistry, Centre for Protein Research, University of Otago, Dunedin, New Zealand.
Torsten
T
Kleffmann
Seino A K
SA
Jongkees
Graham
G
Fairweather
Sigurd M
SM
Wilbanks
Guy N L
GN
Jameson
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D050558:Cysteine Dioxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004583:Electrons; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides; D011499:Protein Processing, Post-Translational; D051381:Rats
D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D050558:Cysteine Dioxygenase