In vivo reconstitution of a homodimeric cytochrome b sub(559) like structure: The role of the N-terminus alpha -subunit from Synechocystis sp. PCC 6803

The cytochrome b sub(559) is a heme-bridged heterodimeric protein with two subunits, alpha and beta . Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the beta -subunit ( beta / beta ) but not with the full length alpha -subunit. In the present work, reconstitution of a homodimer ( alpha / alpha ) cytochrome b sub(559) like structure was possible using a chimeric N-terminus alpha -subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic alpha -helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the alpha -subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric alpha -subunit cytochrome b sub(559) like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b sub(559) maturation in the bacterial cytoplasmic membrane.