Defining the Structural Basis for Assembly of a Transmembrane Cytochrome

Heme binding properties of heterologously expressed spinach cytochrome b6: Implications for transmembrane b‐type cytochrome formation

“…This indicates that the apo-form adopts a structure, which allows binding of the heme molecules and subsequent formation of holo-cytochrome b 6 . This is in good agreement with similar observations made with the transmembrane cytochrome b 559 0 [9,16] and with bacteriorhodopsin [17]. However, the individual steps during cytochrome b 6 formation are still not resolved and it is a major goal of our future work to elucidate the assembly pathway of the transmembrane fourhelix bundle cytochrome b 6 .…”

“…Where indicated, at this step 4 mM succinyl acetone was directly added to the medium to inhibit heme biosynthesis in E. coli. Inclusion bodies and membranes were prepared as described in [9].…”

In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α -subunit from Synechocystis sp. PCC 6803

“…In vivo reconstitution was also possible within the E. coli cytoplasmic membrane overexpressing only the β-subunit from the cyanobacterium Synechocystis sp. PCC 6803, confirming previous in vitro results from chemically synthesized β-subunit and free heme (Franke et al 1999) and in vivo results in recombinant E. coli cells (Prodöhl et al 2005). However, in vivo reconstitution overexpressing only the cyanobacterial α-subunit was unsuccessful, despite that subunit was actually incorporated and stabilized within the bacterial membrane (Luján et al 2012;Luján 2009).…”

“…2b, lane 3). By contrast considering the high structural homology between both subunits, the situation with the full length β-subunit was completely different where the in vivo formation of a Cyt b 559 like structure was indeed possible (Prodöhl et al 2005;Luján et al 2012). According to the structure of the natural heterodimeric (α/β) Cyt b 559 from cyanobacteria ( Figs.…”

“…As a consequence, the MBP protein cannot cross the cytoplasmic membrane to the periplasmic space and thus remains in the cytoplasm. The topology of the MBP protein expressed with these vector series has been examined extensively and unambiguously demonstrated that is located in the periplasmic space when the signal sequence is complete (pMAL-p2) but it appears in the cytoplasm when the signal sequence has the above mentioned deletion (pMAL-c2X) (Prodöhl et al 2005;Kroliczewski et al 2005;Dreher et al 2007;Weber and Schneider 2013).…”