Cytochrome c M from Synechocystis 6803

Abstract

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 m m −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.