Biochemical and mutational characterization of the heme chaperone CcmE reveals a heme binding site.
CcmE is a heme chaperone that binds heme transiently in the periplasm of Escherichia coli and delivers it to newly synthesized and exported c-type cytochromes. The chemical nature of the covalent bond between heme and H130 is not known. We have purified soluble histidine-tagged CcmE and present its spectroscopic characteristics in the visible range. Alanine scanning mutagenesis of conserved amino acids revealed that H130 is the only residue found to be strictly required for heme binding and delivery. Mutation of the hydrophobic amino acids F37, F103, L127, and Y134 to alanine affected CcmE more than mutation of charged and polar residues. Our data are in agreement with the recently solved nuclear magnetic resonance structure of apo-CcmE (PDB code 1LIZ) and suggest that heme is bound to a hydrophobic platform at the surface of the protein and then attached to H130 by a covalent bond. Replacement of H130 with cysteine led to the formation of a covalent bond between heme and C130 at a low level. However, the H130C mutant CcmE was not active in cytochrome c maturation. Isolation and characterization of the heme-binding peptides obtained after a tryptic digest of wild-type and H130C CcmE support the hypothesis that heme is bound covalently at a vinyl group.
Enggist
Elisabeth
E
Departement Biologie, Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Schneider
Michael J
MJ
Schulz
Henk
H
Thöny-Meyer
Linda
L
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003574:Cytochrome c Group / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D018832:Molecular Chaperones; D017354:Point Mutation
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; C115384:CcmE protein, E coli; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D018832:Molecular Chaperones; D006418:Heme