Open AccessCytochrome c M from Synechocystis 6803
Author(s) -
Cho Yoon Shin,
Pakrasi Himadri B.,
Whitmarsh John
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01092.x
Subject(s) - heme , cytochrome , synechocystis , cytochrome c , histidine , cytochrome b , chemistry , isoelectric point , hemeprotein , cytochrome c1 , biochemistry , amino acid , coenzyme q – cytochrome c reductase , stereochemistry , mutant , enzyme , mitochondrion , gene , mitochondrial dna
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.