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

Abstract

SUMMARY Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.

A heme fusion tag for protein affinity purification and quantification

“…The heme tag is a small peptide fusion tag that binds heme covalently to a protein on expression in Escherichia coli and has been used for visual tracking of protein expression and purification. 7 This tagging strategy takes advantage of the bacterial cytochrome c (cyt c) maturation (ccm) apparatus in E. coli, 8 which covalently adds the cysteines of a conserved Cys-X-X-Cys-His (CXXCH) motif to the two heme vinyl groups. 9 The ccm system is composed of eight integral membrane proteins (ccmABCDEFGH) that face the periplasmic side of the cytoplasmic membrane.…”

“…9 The ccm system is composed of eight integral membrane proteins (ccmABCDEFGH) that face the periplasmic side of the cytoplasmic membrane. 8 Thus, proteins carrying the peptide fusion must contain an aminoterminal signal sequence for translocation to the periplasmic space for covalent heme attachment. 7 A complete understanding of the mechanism by which the cofactor is attached to the CXXCH motif by the ccm system is an active area of research.…”

“…7 A complete understanding of the mechanism by which the cofactor is attached to the CXXCH motif by the ccm system is an active area of research. 8 However, it has been shown that recognition for heme attachment depends on the presence of a CXXCH motif. 9 In this study, we make use of heme coordination chemistry and demonstrate that proteins tagged with five-coordinate heme will reversibly bind L-histidine immobilized to a sepharose framework, resulting in a highly pure protein sample after elution.…”

CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation

“…During Ccm, heme binding site Cys-thiols are thought to undergo oxidation reduction via the periplasmic thio-redox pathways (5,6,23). Any requirement for a defined redox state of the heme binding site Cys residues for CcmI-apocytochrome c 2 interaction was tested by DTT/IAM treatments of native and mutated apocytochrome c 2 proteins.…”

“…It is a complex process that involves up to 10 membrane proteins: CcmABCDEFGHI and CcdA (5,6). These components are responsible for (i) transport and relay of heme (CcmABCDE), (ii) preparation and chaperoning of ligation-competent apocytochromes c (CcdA and CcmG), and (iii) ligation of heme to apocytochromes c (Ccm-FHI) to produce mature holocytochromes c (5,6). The final step of this intricate process is the ligation per se of heme b to apocytochrome c to form mature cytochrome c, and this is attributed to the multisubunit membrane complex CcmFHI (7) (see Fig.…”