Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.
To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.
Jiang
Yongying
Y
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA 94158-2517, United States.
Trnka
Michael J
MJ
Medzihradszky
Katalin F
KF
Ouellet
Hugues
H
Wang
Yongqiang
Y
Ortiz de Montellano
Paul R
PR
D001059:Apoproteins / Q000302:isolation & purification / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*
D001059:Apoproteins; D008024:Ligands; D017279:Selenocysteine; D006418:Heme; D007501:Iron; D051547:Heme Oxygenase-1