Multiple step assembly of the transmembrane cytochrome b6.
We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b(6), and we show that the two hemes b(L) and b(H) bind in two subsequent steps to the apo-protein. Binding of the low-potential heme b(L) is a prerequisite for binding the high-potential heme b(H). After substitution of His86, which serves as an axial ligand for heme b(L), the apo-protein did not bind heme, while substitution of the heme b(L)-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme b(H), binding of only heme b(L) was observed, whereas replacement of His100, the other heme b(H) ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme b(L) or heme b(H)), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b(6) variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b(6).
Dreher
Carolin
C
Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, Stefan-Meier-Strasse 17, 79104 Freiburg, Germany.
Prodöhl
Alexander
A
Hielscher
Ruth
R
Hellwig
Petra
P
Schneider
Dirk
D
D045347:Cytochromes b6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018724:Spinacia oleracea / Q000737:chemistry
D045347:Cytochromes b6; D010940:Plant Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D006418:Heme; D006639:Histidine