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

Abstract

In vivo and in vitro requirements for the formation of cytochrome b 6 were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b 6 , formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b 6 appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b 6 was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.

Multiple Step Assembly Of The Transmembrane Cytochrome b6

“…coli, none of the variants was integrated into the E. coli inner membrane anymore as efficiently as the wt, although the proteins were still highly expressed and accumulated in inclusion bodies. While we observed a distinct cytochrome b 6 signal in E. coli membranes after expression of the wt protein, 29 we detected only E. coli endogenous cytochromes after expression of any of the variants. This observation suggests strongly that assembly and stability of cytochrome b 6 variants is somehow affected and can, therefore, not be studied appropriately within a membrane.…”

“…Additionally, the heterologously expressed apo-protein can be purified, and addition of free heme results in spontaneous formation of holo-cytochrome b 6 in vitro. 29,30 Since cytochrome b 6 can be expressed easily in E. coli, and the holo-protein assembles spontaneously from the apo-protein and free heme, cytochrome b 6 can serve as an excellent model for following folding, assembly, and stability of a transmembrane b-type cytochrome in particular and of a cofactor containing membrane protein in general.…”

“…Furthermore, membrane integration and heme binding are most likely not coupled, since expression of apo-cytochrome b 6 under heme depletion conditions did not affect membrane integration. 29 The holo-cytochrome assembled after addition of free heme to these membranes. Additionally, the heterologously expressed apo-protein can be purified, and addition of free heme results in spontaneous formation of holo-cytochrome b 6 in vitro.…”

SDS-Facilitated In vitro Formation of a Transmembrane B-Type Cytochrome Is Mediated by Changes in Local pH

“…Recently, we introduced the model systems cytochrome b 6 and cytochrome b 559 ′, which allow to study binding of heme cofactor to a TM apo-cytochrome. [11][12][13][14][15][16][17] Cytochrome b 559 ′ is a homodimer of the 44-residue-long PsbF polypeptide, which spans the membrane with a single TM helix. One heme cofactor is non-covalently attached to the protein, and the heme iron is ligated to the protein via two histidine residues of the two TM helices, which act as fifth and sixth ligands, respectively.…”

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

“…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).…”