Recombinant cytochromes <i>c</i> biogenesis systems I and II and analysis of haem delivery pathways in <i>Escherichia coli</i>

Feissner, Robert E.; Richard-Fogal, Cynthia L.; Frawley, Elaine R.; Loughman, Jennifer A.; Earley, Keith; Kranz, Robert G.

doi:10.1111/j.1365-2958.2006.05132.x

Cited by 83 publications

(154 citation statements)

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“…For these preliminary studies, nonhexahistidinetagged cyt c variants were used. Cyt c variants were induced with 0.16% arabinose and HCCS W118A, N155A, or E159A with isopropyl-β-D-thiogalactopyranoside (IPTG) in E. coli deleted of its own cyt c biogenesis pathway (MG1655 Δccm) (10). Cyt c levels were quantified from 1-L culture supernatants after ion exchange chromatography.…”

Section: Resultsmentioning

confidence: 99%

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Mendez

Babbitt

King

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c. In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli. Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; −19 mV, M81A; −69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these “release mutants.”

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Section: Resultsmentioning

confidence: 99%

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Mendez

Babbitt

King

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…This ORF (from Helicobacter), when overexpressed in E. coli deleted of its ccm (system I) genes, was all that was necessary to synthesize a periplasmic cytochrome c (57). In this recombinant reconstitution, the CXXCH motif is reduced by the housekeeping reductants (e.g., DsbD and DsbC [E. R. Frawley and R. G. Kranz, unpublished data]).…”

Section: System Ii: Ccsb and Ccsa Form A Heme Channel And Cytochrome mentioning

confidence: 99%

“…A second predicted function of CcmF is binding to holoCcmE as part of its synthetase activity, using the holoCcmE heme for attachment to apocytochrome c (57,123,125). The CcmF (H173A) protein has wild-type cytochrome b function (125) yet is completely nonfunctional (123,125).…”

Section: System I: Ccmabcdefghmentioning

confidence: 99%

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

Kranz¹,

Richard-Fogal²,

Taylor

et al. 2009

Microbiol Mol Biol Rev

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235

372

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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.

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“…In principle, the chaperone-like action could be an interaction between PecE and PCB by which the latter delivered in the correct conformation to PecA; a (mechanistically different, see below) preparatory action is also discussed for the "chaperone," CcmE in cytochrome c formation (30). Several lines of evidence, however, suggest that prior formation of complexes between PecA and PecE accelerates this action.…”

Section: Protein-protein Interactionsmentioning

confidence: 99%

“…System III involves the CchL-type heme-lyases to doubly link the heme chromophore to the apo-cytochromes (63)(64)(65)(66). The other systems involve several other proteins, including heme-transporters ("chaperones"), and, only recently, has the lyase function been assigned to a pair of gene products, CcsA and CcsB (30). Again, no obvious homologies are so far apparent for either the phytochrome lyase domains or the phycobiliproteins lyases, even though the apoproteins of cytochromes are phylogenetically and structurally related to those of the biliproteins (35).…”

Section: Assignment Of Intermediatesmentioning

confidence: 99%

Biliprotein Chromophore Attachment

Böhm

Endres

Scheer

et al. 2007

Journal of Biological Chemistry

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Biliproteins are post-translationally modified by chromophore addition. In phycoerythrocyanin, the heterodimeric lyase PecE/F covalently attaches phycocyanobilin (PCB) to cysteine-␣84 of the apoprotein PecA, with concomitant isomerization to phycoviolobilin. We found that: (a) PecA adds autocatalytically PCB, yielding a low absorbance, low fluorescence PCB⅐PecA adduct, termed P645 according to its absorption maximum; (b) In the presence of PecE, a high absorbance, high fluorescence PCB⅐PecA adduct is formed, termed P641; (c) PecE is capable of transforming P645 to P641; (d) When in stop-flow experiments, PecA and PecE were preincubated before chromophore addition, a red-shifted intermediate (P650, ‫؍‬ 32 ms) was observed followed by a second, which was blue-shifted (P605, ‫؍‬ 0.5 s), and finally a third (P638, ‫؍‬ 14 s) that yielded the adduct (P641, ‫؍‬ 20 min); (e) The reaction was slower, and P605 was missing, if PecA and PecE were not preincubated; (f) Gel filtration gave no evidence of a stable complex between PecA and PecE; however, complex formation is induced by adding PCB; and (g) A red-shifted intermediate was also formed, but more slowly, with phycoerythrobilin, and denaturation showed that this is not yet covalently bound. We conclude, therefore, that PecA and PecE form a weak complex that is stabilized by PCB, that the first reaction step involves a conformational change and/or protonation of PCB, and that PecE has a chaperone-like function on the chromoprotein.Phycobiliproteins are a family of light-harvesting proteins of cyanobacteria, rhodophytes, and cryptophytes (1-4). The apoproteins are post-translationally modified by regio-and stereoselective chromophore attachment to cysteines. Addition to the cysteine-␣84 of cyanobacterial phycocyanins and phycoerythrins is catalyzed by the heterodimeric E/F-type lyases (5). The first enzyme of this type studied, phycocyanobilin:cysteine-␣84-phycocyanin lyase, from the cyanobacterium, Synechococcus sp. PCC 7002, attaches phycocyanobilin (PCB) 3 to apo-␣-phycocyanin (CpcA) (6). It is encoded by two genes, cpcE and cpcF, located on the phycocyanin (CPC) operon downstream of the two apo-phycocyanin and two linker genes. A similar operon structure has been found in several other organisms, but the respective genes can also be located in other positions (5, 7). The functions of the two subunits are still unclear. Chromophore attachment to the phylogenetically related site on the ␤-subunit, cysteine-␤82, is catalyzed by a single-subunit protein, or mixtures of similar proteins (8, 9), whereas the chromophores are attached autocatalytically in phytochromes (10, 11), allophycocyanin, ApcA (12), and the core-membrane linker, ApcE (13). The situation is complicated by the capacity of apo-phycobiliproteins like CpcA/B to also add chromophores autocatalytically but only slowly and in an error-prone fashion (14 -17).The isomerizing phycoviolobilin:cysteine-␣84-phycoerythrocyanin lyase is a member of the E/F-type lyases, which, in addition to chromophore attachment, ha...

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Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli

Cited by 83 publications

References 55 publications

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

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

Biliprotein Chromophore Attachment

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