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ABSTRACT: The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
Biochimica et Biophysica Acta 09/2011; 1817(6):911-9. · 4.66 Impact Factor
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ABSTRACT: A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe(3+)), yet the final attachment requires reduced haem (Fe(2+)). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.
The EMBO Journal 08/2009; 28(16):2349-59. · 9.20 Impact Factor
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ABSTRACT: The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.
Journal of bacteriology 06/2008; 190(10):3489-93. · 3.94 Impact Factor
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ABSTRACT: Studies have indicated that specific heme delivery to apocytochrome c is a critical feature of the cytochrome c biogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromes c, we engineered Escherichia coli so that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (Delta hemA). The engineered E. coli strains that produced recombinant system I (from E. coli) or system II (from Helicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromes c with other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II).
Journal of Bacteriology 02/2007; 189(2):455-63. · 3.83 Impact Factor
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ABSTRACT: Although organisms from all kingdoms have either the system I or II cytochrome c biogenesis pathway, it has remained a mystery as to why these two distinct pathways have developed. We have previously shown evidence that the system I pathway has a higher affinity for haem than system II for cytochrome c biogenesis. Here, we show the mechanism by which the system I pathway can utilize haem at low levels. The mechanism involves an ATP-binding cassette (ABC) transporter that is required for release of the periplasmic haem chaperone CcmE to the last step of cytochrome c assembly. This ABC transporter is composed of the ABC subunit CcmA, and two membrane proteins, CcmB and CcmC. In the absence of CcmA or CcmB, holo(haem)CcmE binds to CcmC in a stable dead-end complex, indicating high affinity binding of haem to CcmC. Expression of CcmA and CcmB facilitates formation of the CcmA2B1C1 complex and ATP-dependent release of holoCcmE. We propose that the CcmA2B1C1 complex represents a new subgroup within the ABC transporter superfamily that functions to release a chaperone.
Molecular Microbiology 08/2006; 61(1):219-31. · 5.01 Impact Factor
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ABSTRACT: Genetic analysis has indicated that the system II pathway for c-type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA-H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c-haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N-methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo-CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo-CcmE as a haem reservoir, a capability system II does not possess.
Molecular Microbiology 06/2006; 60(3):563-77. · 5.01 Impact Factor
