Dynamic Ligation Properties of the Escherichia coli Heme Chaperone CcmE to Non-covalently Bound Heme

Abstract

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.c-type cytochromes are proteins that contain covalently attached heme cofactors and are essential for the life of virtually all organisms. Cytochrome c in the mitochondria respiratory chain contains a single heme molecule, whereas many bacterial c-type cytochromes are found to contain many hemes; a Geobacter cytochrome c appears to contain 27 heme molecules (1). The heme is characteristically covalently attached to a CXXCH motif and allows the proteins to function in electron transfer and to catalyze a variety of reactions. The biogenesis of c-type cytochromes is a complex post-translational process that, in Gram-negative bacteria such as Escherichia coli, occurs in the periplasm. The heme attachment reaction appears to occur by very different pathways in different types of organisms; in humans, for example, a single protein called heme lyase is involved, whereas in E. coli at least ten proteins are essential for the process (2, 3). The latter system is known as the cytochrome c maturation (Ccm) 6 system and comprises the proteins CcmABCDEFGH, all of which are anchored to the cytoplasmic membrane or integral to it (4) as indicated in Fig. 1a. The membrane-anchored protein CcmE has been described as a heme chaperone and binds heme covalently in the bacterial periplasm before the heme is transferred to apocytochromes (5). Unusually, the protein binds heme covalently via a single histidine residue, as opposed to the binding of heme to two cysteine residues in cytochromes c. It is not known how heme is transported into the periplasm from its site of synthesis in the cytoplasm, but it is known that the protein CcmC is essential for the provision of heme to CcmE (6). The protein CcmF has been shown to form a complex with CcmE (7), but its function remains unknown, as do the precise roles of the other Ccm proteins. Recently, CcmD was sho...

The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme-Binding Protein

“…The 1470 cm -1 band is characteristic of the 5-coordinate high-spin ferrous heme. The frequency of the 1499 cm -1 band was slightly higher than that of the typical 6-coordinate low-spin heme (~1490 cm -1 ), but generally similar to that observed for tyrosine-coordinated ferrous heme, 31,38,40 indicating that ferrous heme-CyaY contains a 4-coordinate heme, not a 6-coordinate heme. The position of the frequencies of ν Fe-CO and ν C-O on the correlation plot between ν Fe-CO and ν C-O provides useful insight into the donor strength of the trans ligand of iron-bound CO. 41 In the spectrum of the CO-bound form, two isotope-sensitive bands appeared at 495 and 1965 cm -1 , which shifted to 485 and 1873 cm -1 , respectively, upon 13 C 18 O replacement (Supplemental Figure S3).…”

The CcmC:Heme:CcmE Complex in Heme Trafficking and Cytochrome c Biosynthesis

“…We previously demonstrated that formation of the CcmC:heme: CcmE complex does not require the CcmE His130 covalent adduct, although the split α absorption is perturbed in the CcmE(H130A) mutant and the heme is not covalently attached to CcmE. 16 Some residues in CcmE have been studied for the in vitro binding of free heme to mutated soluble CcmE*, 13,15,45 but the relevance to the interactions in formation of the complex described here is unknown. An NMR-derived structure for apoCcmE* has been determined, 42,45 and prior models have speculated on where heme might reside in the released holoCcmE.…”

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.…”

“…The fact that heme in the preadduct CcmCDE complex is approximately 50% reduced (125) could argue for either mechanism. The in vitro synthesis of the holoCcmE* adduct appears to require the reductant dithionite (15,42,148), leading to the suggestion of a radical mechanism, although it is unclear whether a cation or an anion radical is proposed. The mechanism by which this radical might be formed in vitro or in vivo is still a mystery, but note that with the cation radical, the end product has oxidized (Fe 3ϩ ) heme.…”