Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

Abstract

A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

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

“…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). Thus the heme group was not necessary for the incorporation and stabilization of the Cyt b 559 α-subunit within the membrane.…”

“…In a previous publication (Luján et al 2012), we described the in vitro reconstitution of a Cyt b 559 like structure using the recombinant subunits from the plant sugar beet overexpressed in Escherichia (E.) coli and partially purified from inclusion bodies. Thus homodimeric and heterodimeric Cyt b 559 like structures were reconstituted using commercial heme with a yield somewhat higher for the heterodimer (α/β) form, the natural structure found in the PSII, than for the homodimers (α/α; β/β) (Luján et al 2012).…”

“…Thus homodimeric and heterodimeric Cyt b 559 like structures were reconstituted using commercial heme with a yield somewhat higher for the heterodimer (α/β) form, the natural structure found in the PSII, than for the homodimers (α/α; β/β) (Luján et al 2012). In vivo reconstitution was also possible within the E. coli cytoplasmic membrane overexpressing only the β-subunit from the cyanobacterium Synechocystis sp.…”

“…The full length psbE gene from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed as in Luján (2009) and Luján et al (2012). A chimeric Cyt b 559 α-subunit was generated by removing the first sixteen amino acid residues of the Nterminus, thus the new chimeric protein sequence began at the isoleucine 17 (called thereafter SsubαI17 or αI17).…”

“…Protein overexpression in E. coli TB1 competent cells was described in detail in Luján (2009) and Luján et al (2012). Essentially, the overexpression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 ºC for 3 h or 1 mM IPTG at 18 ºC for 5 h. Cytoplasmic membrane fragments were obtained according to the protocol published in the aforementioned paper.…”