Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase.
Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.
Strittmatter
P
P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
Hackett
C S
CS
Korza
G
G
Ozols
J
J
D000577:Amides; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D003579:Cytochrome Reductases / Q000737:chemistry* / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation
D000577:Amides; D003432:Cross-Linking Reagents; D010455:Peptides; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D008239:Lysine