The Heme-Containing Portion of Cytochrome c _z from Chlorobium tepidum: Its Over-Expression in Escherichia coli and Spectroscopic Studies

Abstract

Green sulfur bacteria have the type 1 reaction center (RC) similar to photosystem I in cyanobacteria and higher plants. Cytochrome c _z (cyt c _z) contains a heme c and serves as a direct electron donor to the P840. It is anchored to membranes with its N-terminal hydrophobic helices and holds a heme in its C-terminal hydrophilic domain. As the electron transfer rate strongly depends on the viscosity of reaction media, the heme-containing moiety is considered to fluctuate all the time while searching for the appropriate reaction surface on the RC complex. The C-terminal hemecontaining portion (C-cyt c _z) was over-expressed in Escherichia (E.) coli in order to study its reaction characteristics in detail. We constructed a co-expression system containing a plasmid pEC86 that carries the cytochrome c maturation genes cluster derived from E. coli to express the hemeattached C-cyt c _z. The C-cyt c _z was expressed in the periplasmic space of E. coli strain C41 and purified with ion-exchange and gel filtration chromatographies. The expressed C-cyt c _z showed typical absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra of c-type cytochromes. But ¹H-nuclear magnetic resonance (NMR) spectrum of the oxidized form revealed larger paramagnetic shifts in low-field compared with those of other low-spin c-type cytochromes. This suggests that the conformation around the heme group is different from the other c-type cytochromes.