Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme.
The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.
Stevens
Julie M
JM
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Uchida
Takeshi
T
Daltrop
Oliver
O
Kitagawa
Teizo
T
Ferguson
Stuart J
SJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; C115384:CcmE protein, E coli; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008024:Ligands; D018832:Molecular Chaperones; D006418:Heme