Cytochrome cM 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 cM, 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 cM in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome cM 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 alpha band at 550 nm. The extinction coefficient of cytochrome cM is 23.2 +/- 0.5 mM-1.cm-1 for the reduced minus oxidized alpha band peak (550-535 nm). The isoelectric point of cytochrome cM 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 cM 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 cM and investigating its function in cyanobacteria.
Cho
Y S
YS
Department of Plant Biology, Photosynthesis Research Unit, USDA/ARS, University of Illinois, Urbana, USA.
Pakrasi
H B
HB
Whitmarsh
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015153:Blotting, Western; D000458:Cyanobacteria / Q000201:enzymology*; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007526:Isoelectric Point; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D012995:Solubility; D013057:Spectrum Analysis; D017186:Titrimetry
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011993:Recombinant Fusion Proteins; C117990:cytochrome c(M), Synechocystis; D006418:Heme; D006639:Histidine; D008715:Methionine