The CcmE protein of the c-type cytochrome biogenesis system: unusual in vitro heme incorporation into apo-CcmE and transfer from holo-CcmE to apocytochrome.
Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro. apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (K(d), 200 nM), rather than ferrous heme. The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE. Reduction under certain conditions of the ferric heme-CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein. The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE. Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE. Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced. The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products. In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE. These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Oliver
O
Daltrop
Julie M
JM
Stevens
Christopher W
CW
Higham
Stuart J
SJ
Ferguson
D001051:Apoenzymes / Q000096:biosynthesis; D001059:Apoproteins / Q000096:biosynthesis* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000378:metabolism*; D007700:Kinetics
D001051:Apoenzymes; D001059:Apoproteins; D001425:Bacterial Outer Membrane Proteins; C115384:CcmE protein, E coli; D003574:Cytochrome c Group; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D006418:Heme