The role of cysteine residues in the oxidation of ferritin.
We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999-1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates.
Welch
Kevin D
KD
Biotechnology Center, Utah State University, Logan, UT 84322-4705, USA.
Reilly
Christopher A
CA
Aust
Steven D
SD
D002499:Centrifugation, Density Gradient; D003497:Cyclic N-Oxides; D003545:Cysteine / Q000502:physiology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000502:physiology; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives*
D003497:Cyclic N-Oxides; D010446:Peptide Fragments; D013113:Spin Labels; D014443:Tyrosine; D005293:Ferritins; C007543:dityrosine; D014357:Trypsin; D003545:Cysteine; C003959:TEMPO