Modification by homocysteine thiolactone affects redox status of cytochrome C.
Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.
Perła-Kaján
Joanna
J
Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznań, Poland. kajan@au.poznan.pl
Marczak
Łukasz
Ł
Kaján
László
L
Skowronek
Paweł
P
Twardowski
Tomasz
T
Jakubowski
Hieronim
H
D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D006736:Horses; D020138:Hyperhomocysteinemia; D008239:Lysine / Q000378:metabolism; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary
D006710:Homocysteine; D045304:Cytochromes c; C007957:homocysteine thiolactone; D008239:Lysine