Stereoselective in vitro formation of c-type cytochrome variants from Hydrogenobacter thermophilus containing only a single thioether bond.
In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). Now we report that the single cysteine variants of H. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality. This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation. Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H. thermophilus c552 to yield another modification of cytochromes containing only one thioether bond. These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another. Aspects of rotational isomerism of heme in heme-proteins are discussed.
Daltrop
Oliver
O
Department of Biochemistry, University of Oxford, South Parks Road, United Kingdom.
Smith
Kevin M
KM
Ferguson
Stuart J
SJ
D001420:Bacteria, Aerobic / Q000201:enzymology; D003545:Cysteine; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation
D003574:Cytochrome c Group; D006418:Heme; D003545:Cysteine