Cytochrome c(M) from Synechocystis 6803: Detection in cells, expression in Escherichia coli, purification and physical characterization

Based on DNA sequence data a novel c-type cytochrome, cytochrome c(M), has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme- binding motif and a hydrophobic domain located at the N-terminal end that is proposed to act as either a signal peptide or a membrane anchor. For the first time we report the detection of cytochrome c(M) in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome c(M) has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag. The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the α band at 550 nm. The extinction coefficient of cytochrome c(M) is 23.2 ± 0.5 mM^-1·cm^-1 for the reduced minus oxidized α band peak (550-535 nm). The isoelectric point of cytochrome c(M) is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence. The redox midpoint potential of cytochrome c(M) expressed in E. coli is 151 ± 5 mV (pH 7.1), which is quite low compared to other c-type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three-dimensional structure of cytochrome c(M) and investigating its function in cyanobacteria.