Physico-chemical characterisation of membrane-bound and water-soluble forms of Bacillus subtilis cytochrome c-550.
Cytochrome c-550 of the Gram-positive bacterium, Bacillus subtilis, is a membrane-bound 13-kDa protein encoded by the cccA gene. The cytochrome has been proposed to be comprised of an N-terminal membrane anchor domain (about 30 residues) which spans the cytoplasmic membrane in an alpha-helical conformation and a C-terminal heme domain (about 70 residues) which is located on the outside of the cytoplasmic membrane. Cytochrome c-550 was purified in the presence of Triton X-100 and characterised. In the reduced state it shows absorption maxima at 415, 521, 550 nm and in the oxidised state a Soret band at 408 nm and a weak band at about 695 nm. The latter absorption band, together with data from amino acid sequence comparisons, strongly suggest His64 and Met99 as the fifth and sixth axial ligands to the heme iron in cytochrome c-550. The midpoint redox potential of the cytochrome, +178 mV, was pH-independent in the pH range 6.0-7.9. Oxidised cytochrome c-550 showed an EPR signal at gmax = 3.41, which is unusual for low-spin cytochromes c with His/Met axial ligation. The heme domain was isolated as a tryptic fragment of 74 residues and as a protein-A-cytochrome-c-550 hybrid protein. Both these forms were water-soluble and showed thermodynamic and spectroscopic properties indistinguishable from the membrane-bound form of cytochrome c-550 and are suitable for structural analysis of the heme domain by X-ray crystallography or NMR techniques. Polypeptide analysis of the membrane-bound and water-soluble tryptic fragment confirmed that B. subtilis cytochrome c-550 in the membrane consists of 120 amino acid residues and has a two-domain structure.
Department of Microbiology, University of Lund, Sweden.
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von Wachenfeldt
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Hederstedt
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003574:Cytochrome c Group / Q000737:chemistry* / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D008969:Molecular Sequence Data; D011199:Potentiometry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D012995:Solubility; D013053:Spectrophotometry
D003574:Cytochrome c Group; D011993:Recombinant Fusion Proteins; C031732:cytochrome C-550