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

Abstract

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 hemeCcmE 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.

The Interaction of Covalently Bound Heme with the Cytochrome c Maturation Protein CcmE

“…Purified CcmE produced under the conditions described contained a substantial amount of apo-protein; usually about 20% of the protein contained covalently attached heme as determined by comparing the ratio of the absorbance value at 280 nm (apoprotein ϩ holo-protein) with the intensity of the Soret band (holo-protein). The co-production of large amounts of apoprotein with holo-protein has been observed previously with CcmE (6,11). Only the apo-forms of the proteins produced were observed in the mass spectra; the heme-containing form does not appear in the spectra under these conditions.…”

“…Cultures were first grown to mid-log phase at 37°C following inoculation and then induced with 1 mM isopropyl-thio-␤-D-galactopyranoside and grown overnight at 28°C. Proteins were purified as described previously (11). Thrombin digestion was performed using a CleanCleave kit (Sigma) according to the manufacturer's instructions, and the uncleaved protein was separated from the cleaved protein by reapplying the protein mixture to a Ni 2ϩ -chelating Sepharose column.…”

“…Protein concentrations were determined using the Bradford method (Bio-Rad), and the protein was stored in 50 mM Tris-HCl, pH 7.4, 300 mM NaCl throughout. The protein referred to in this work as CcmE refers to the soluble form of CcmE that lacks its membrane anchor, as described (11).…”

“…The apparent conformational flexibility of the protein may explain why crystallization of CcmE has not been reported. Therefore, the only information at present on the nature of the heme ligation in holo-CcmE is limited to UV-visible spectra (6,11,12), which suggest that the heme adopts a high spin state in the ferric form and a 6-coordinate low spin state in the ferrous form. The difference in the heme ligation between the two redox states suggests a working hypothesis for the function of the protein, particularly regarding the control of heme binding and release according to the redox state of the heme iron.…”

AtCCMA Interacts with AtCcmB to Form a Novel Mitochondrial ABC Transporter Involved in Cytochrome c Maturation in Arabidopsis

“…However, in phylogenetic analysis, the CcmA family cluster belongs to the exporter sub family ABC-A2, which contains proteins having the ABC modules not fused to TMD (46). An electron donor, essential to maintain the heme iron in a reduced state after its transfer to CcmE, could be a possible substrate for the system I ABC transporter and essential for the release of heme to apocytochrome c. Indeed, in vitro and in vivo approaches showed that heme iron needs to be reduced for the covalent linkage to occur (47)(48)(49). In yeast mitochondria, a flavin-linked electron transfer was postulated for heme lyase reaction (49), and Cyc2p a flavoprotein was proposed to reduce heme iron before its attachment to apocytochrome c by cytochrome c heme lyase (50).…”