Characterization of Hydrogenobacter thermophilus cytochromes c(552 )expressed in the cytoplasm and periplasm of Escherichia coli.
Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).
Karan
Elizabeth F
EF
Department of Chemistry, University of Rochester, Rochester, NY 14627-0216, USA.
Russell
Brandy S
BS
Bren
Kara L
KL
D001420:Bacteria, Aerobic / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D019906:Nuclear Magnetic Resonance, Biomolecular; D019897:Periplasm / Q000201:enzymology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D003574:Cytochrome c Group; C083951:cytochrome c553; C022302:cytochrome C-552