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 c 552 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 c 552 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.
Publication Source (Journal or Book title)
Journal of Biological Chemistry
Daltrop, O., Smith, K., & Ferguson, S. (2003). Stereoselective in Vitro Formation of c-type Cytochrome Variants from Hydrogenobacter thermophilus Containing only a Single Thioether Bond. Journal of Biological Chemistry, 278 (27), 24308-24313. https://doi.org/10.1074/jbc.M301967200