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 precu rsor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic domain located at the N-terminal end that is propo sed to act as either a signal peptide or a membrane anchor. For the first t ime we report the detection of cytochrome c(M) in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome c(M) has been overexp ressed in Escherichia coli in two forms, one with a poly histidine tag to f acilitate purification and one without such a tag. The overexpressed protei n has been purified and shown to bind heme, exhibiting an absorption peak i n the Sent band near 416 nm and a peak in the alpha band at 550 nm. The ext inction coefficient of cytochrome c(M) is 23.2 +/- 0.5 mM(-1).cm(-1) for th e reduced minus oxidized alpha band peak (550-535 nm). The isoelectric poin t of cytochrome c(M) is 5.6 (without the histidine tag), which is significa ntly lower than the pI of 7.2 predicted from the amino acid sequence. The r edox 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 w hich a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three-dimensional struct ure of cytochrome c(M) and investigating its function in cyanobacteria.