In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

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

“…While our in vitro observations, which are in good agreement with the in vivo data shown in Fig. 3, suggest that apocytochrome b 6 adopts a folding state, which is highly competent for heme binding, the observations in [14] suggest that folding of cytochrome b 6 depends on the presence of heme and thus heme binding would be an early step in the folding pathway of holo-cytochrome b 6 . While this discrepancy cannot be completely resolved yet, in [14] cytochrome b 6 was formed very slowly in vitro and the added heme needed to be stable for several hours.…”

“…3, suggest that apocytochrome b 6 adopts a folding state, which is highly competent for heme binding, the observations in [14] suggest that folding of cytochrome b 6 depends on the presence of heme and thus heme binding would be an early step in the folding pathway of holo-cytochrome b 6 . While this discrepancy cannot be completely resolved yet, in [14] cytochrome b 6 was formed very slowly in vitro and the added heme needed to be stable for several hours. Air oxygen can rapidly oxidize heme and the molecule is not very stable for a long time, which could explain that in [14] reconstitution was only efficient under reducing conditions.…”

“…Heme can be incorporated into a membrane bound apocytochrome It has recently been suggested that the time point of heme addition is critical for holo-cytochrome b 6 formation within the E. coli inner membrane and that the holo-cytochrome forms only when heme is added prior induction of protein expression [14]. Thus, assembly of holo-cytochrome b 6 would depend on the presence of free heme during membrane integration of the apo-protein.…”

“…While this discrepancy cannot be completely resolved yet, in [14] cytochrome b 6 was formed very slowly in vitro and the added heme needed to be stable for several hours. Air oxygen can rapidly oxidize heme and the molecule is not very stable for a long time, which could explain that in [14] reconstitution was only efficient under reducing conditions. Based on our results there is no need for using urea and SDS for holo-cytochrome b 6 formation and cytochrome b 6 can be efficiently reconstituted in a wide variety of detergents (not shown), including the mild detergent b-dodecylmaltoside.…”

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

“…The amount of heme required to reconstitute the remainder of the purified sample was calculated based on the smallscale titration result. To verify that the observed spectral changes were not due to adventitious binding of heme to MBP, hemin chloride was also titrated into free MBP as previously reported [29].…”