The interaction of heme with the heme chaperone CcmE is central to our understanding of cytochrome c maturation, a complex post-translational process involving at least eight proteins in many Gram-negative bacteria and plant mitochondria. We have shown previously that Escherichia coli CcmE can interact with heme non-covalently in vitro, before forming a novel covalent histidine-heme bond, in a redox-sensitive manner. The function of CcmE is to bind heme in the periplasm before transferring it to apocytochromes c. In the absence of structural information on the complex of CcmE and heme, we have further characterized it by examining the binding of the soluble domain of CcmE (CcmE') to protoporphyrins containing metals other than Fe, namely Zn-, Sn-, Co- and Mn-protoporphyrin (PPIX). CcmE' demonstrated no affinity for the Zn- or Sn-containing protoporphyrins and low affinity for Mn(ii)-PPIX. High-affinity, reversible binding was, however, observed for Co(iii)-PPIX, which was highly sensitive to oxidation state as demonstrated by release of the ligand from the chaperone on reduction; no binding to Co(ii)-PPIX was observed. The non-covalent complex of CcmE' and Co(iii)-PPIX was characterized by non-denaturing mass spectrometry. The implications of these observations for the in vivo function of CcmE are discussed.
Bacterial Outer Membrane Proteins Cobalt