Axial Coordination of Heme in Ferric CcmE Chaperone Characterized by EPR Spectroscopy

Abstract

In Escherichia coli cytochrome c maturation requires a set of eight proteins including the heme chaperone CcmE, which binds heme transiently, yet covalently. Several variants of CcmE were purified and analyzed by continuous-wave electron paramagnetic resonance, electron nuclear double resonance, and hyperfine sublevel correlation spectroscopy to investigate the heme axial coordination. Results reveal the presence of a number of coordination environments, two high-spin heme centers with different rhombicities, and at least one low-spin heme center. The low-spin species was shown to be an artifact induced by the presence of available histidines in the vicinity of the iron. Both of the high-spin forms are five-coordinated, and comparison of the spectra of the wild-type CcmE with those of the mutant CcmE(Y134H) proves that the higher-rhombicity form is coordinated by Tyr134. The low-rhombicity (axial) form does not have a histidine residue or a water molecule as an axial ligand. However, we identified exchangeable protons coupled to the iron ion. We propose that the axial form can be coordinated by a carboxyl group of an acidic residue in the flexible domain of the protein. The two species would represent two different conformations of the flexible alpha-helix domain surrounding the heme. This conformational flexibility confers CcmE special dynamic properties that are certainly important for its function.

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…The unique chemistry and structure of this bond to the 2-vinyl ␤ carbon (93) have been discussed (61,93,147,148,158). We discovered that all CcmCDE postadduct complexes and released holoCcmE (see below) contain Fe 3ϩ heme, suggesting that in addition to covalent bond formation in CcmE, there must also be an oxidation step leading to an oxidized holoCcmE.…”

“…Uchida et al used Raman spectroscopy to indicate that one axial ligand in holoCcmE* (truncated with no TMD) is a histidine and the other is the tyrosine 134 discussed above (Fig. 4) (158), but it is not necessary to envision two heme ligands from CcmE immediately switched to two from CcmF and then to the two from cytochrome c. Garcia-Rubio et al used various spectral approaches on holoCcmE* indicating that tyrosine is one axial ligand, but an unknown sixth ligand may exhibit conformational flexibility (61). In any event, it is likely that the WWD domain binds holoCcmE heme, precisely positioning the vinyl side chains (one with the CcmE adduct) to accept the CXXCH motif of the apocytochrome c (see below).…”

c-Type Cytochrome Biogenesis Can Occur via a Natural Ccm System Lacking CcmH, CcmG, and the Heme-binding Histidine of CcmE

“…4A, panel ii). Unfortunately, the available structure only encompasses residues 44 -128, lacking the N-terminal transmembrane helix but also terminating at the proline of the CPSKY motif (where the cysteine is Cys 127 ), thereby excluding the conserved tyrosine that is a heme-iron ligand in E. coli CcmE (42,43). Nevertheless, the ␤-barrel structure bears a striking similarity to that of the E. coli protein despite relatively low sequence identity.…”

Cytochrome c assembly

“…This is unexpected for a chaperone protein and the importance of the covalent but transient linkage remains unknown. CcmE binds heme non-covalently and then covalently in vitro (39) with a tyrosine ligating the heme iron (40,41). The heme-binding region of the protein is flexible (42)(43)(44), as would be expected from its function.…”