Interaction of heme with variants of the heme chaperone CcmE carrying active site mutations and a cleavable N-terminal His tag.
Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form.
Stevens
Julie M
JM
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Daltrop
Oliver
O
Higham
Christopher W
CW
Ferguson
Stuart J
SJ
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics*; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006639:Histidine; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding
D001059:Apoproteins; D001425:Bacterial Outer Membrane Proteins; C115384:CcmE protein, E coli; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D006418:Heme; D006639:Histidine; D045304:Cytochromes c