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Information on EC 4.4.1.17 - Holocytochrome-c synthase and Organism(s) Homo sapiens and UniProt Accession P53701
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4.4.1.17 Holocytochrome-c synthaseIUBMB Comments
In the reverse direction, the enzyme catalyses the attachment of heme to two cysteine residues in the protein, forming thioether links.
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The taxonomic range for the selected organisms is: Homo sapiens
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
system iii, heme lyase, cytochrome c heme lyase, cyc2p, holocytochrome c synthase, holocytochrome c-type synthase, ccsa1, holocytochrome c synthetase, cytochrome c synthase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CCHL
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Cytochrome c heme-lyase
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Cytochrome c synthase
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Holocytochrome c synthetase
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Holocytochrome c-type synthase
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Holocytochrome-C synthase
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Holocytochrome-C-type synthase
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Synthetase, holocytochrome c
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
covalent attachment of heme
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SYSTEMATIC NAME
IUBMB Comments
holocytochrome-c apocytochrome-c-lyase (heme-forming)
In the reverse direction, the enzyme catalyses the attachment of heme to two cysteine residues in the protein, forming thioether links.
CAS REGISTRY NUMBER
COMMENTARY
75139-03-6
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Apocytochrome c + heme
Holocytochrome c
conserved His154 is the key ligand to the heme iron, formation of the enzyme-heme complex serves as the platform for interaction with apocytochrome c, the heme is the central molecule mediating contact between enzyme and apocytochrome c. Conserved His19 of the CXXCH motif in apocytochrome c supplies the second axial ligand to heme in the trapped enzyme-heme-cytochrome c complex. Molecular mechanisms, overview
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Apocytochrome c + heme
Holocytochrome c
the catalytic function of the enzyme depends on its ability to coordinate interactions between its substrates: heme and cytochrome c, four-step model describing enzyme-mediated cytochrome c assembly, identifying conserved histidine residue 154 as an axial ligand to the heme iron, overview. The enzyme contains two heme-binding domains, heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the enzyme-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding, others impact the subsequent release of the holocytochrome c product
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Apocytochrome c + heme
Holocytochrome c
the enzyme attaches heme to wild-type cytochrome, and to mutant cytochromes containing individual cysteine, histidine, and double cysteine of conserved motif CXXCH, but not the mutant with triple cysteine/histidine substitutions, overview. His19 in cytochrome c is important for 1. to provide the second axial ligand to the heme iron in preparation for covalent attachment, 2. to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation, and 3. to aid in release of the holocytochrome c from the enzyme's active site. Substitutions of His19 in cytochrome c to seven other residues (G,A,M,R,K,C,Y) show that only mutant H19M is able to carry out these three roles, albeit at lower efficiencies than the wild-type His19. The histidine in the CXXCH motif acts as an axial ligand to the heme iron
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additional information
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substrate specificity of the human enzyme, overview. Engineering of a bacterial cytochrome c into a robust substrate for the human enzyme, overview
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additional information
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substrate specificity of the human enzyme, overview. Engineering of a bacterial cytochrome c into a robust substrate for the human enzyme, overview
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additional information
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HCCS mediates heme attachment to the N-terminal cysteine in cytochrome c C-terminal variants, but up to 50% of the cytochrome c produced is modified in an oxygen-dependent manner, resulting in a mixed population of cytochrome c. Natural HCCS-mediated heme attachment to C-terminal cytochrome variants likely initiates at the C-terminal cysteine
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
possibly associated with microphtalmia with linear skin defects syndrome
SwissProt
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
the enzyme is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III
physiological function
physiological function
C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase
physiological function
cytochrome c, an essential electron carrier in mitochondria and a critical component of the apoptotic pathway, contains a heme cofactor covalently attached to the protein at a conserved CXXCH motif requiring the mitochondrial protein holocytochrome c synthase
physiological function
for mitochondrial cytochrome c assembly, the enzyme binds heme and apocytochrome c substrate to catalyze a covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1, and subsequently releases holocytochrome c for proper folding to its native structure, mechanism of assembly, overview
physiological function
human cyts c with deletion of a single residues in helix-1, DELTA M13 cyt c, is poorly matured by human HCCS. Although an HCCS-wild-type cytochrome c complex contains two covalent links, HCCS DELTA M13 cytochrome c contains only one thioether attachment. This attachment is to the second thiol of residues C15SQC18H. The serine residue (of CSQCH) would be anchored where the first cysteine should be in DELTA M13 cytochrome c. An engineered cytochrome c with a CQCH motif in the DELTA M13 background is matured at 2-3fold higher levels
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). CCHL_HUMAN
268
0
30602
Swiss-Prot
other Location (Reliability: 3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C15S
the HCCS:cyt c C15S and C18A mutants support the formation of single thioether cross-links between the heme and cyt c at the vinyl position alternate to the mutation
C18A
the HCCS:cyt c C15S and C18A mutants support the formation of single thioether cross-links between the heme and cyt c at the vinyl position alternate to the mutation
E159A
E159D
site-directed mutagenesis of a domain II residue, the mutant shows altered heme binding compared to the wild-type enzyme
E159K
site-directed mutagenesis of a domain II residue, the mutant shows altered heme binding compared to the wild-type enzyme
M130A
site-directed mutagenesis of a domain I residue, the mutant shows altered heme binding compared to the wild-type enzyme
N128A
site-directed mutagenesis of a domain I residue, the mutant shows altered heme binding compared to the wild-type enzyme
N128A/M130A
site-directed mutagenesis of domain I residues, the mutant shows altered heme binding compared to the wild-type enzyme
N155A
site-directed mutagenesis of a domain II residue, the mutant shows altered heme binding compared to the wild-type enzyme
P121A
site-directed mutagenesis of a domain I residue, the mutant shows altered heme binding compared to the wild-type enzyme
W118A
Y120A
site-directed mutagenesis of a domain I residue, the mutant shows altered heme binding compared to the wild-type enzyme
Y120A/P121A
site-directed mutagenesis of domain I residues, the mutant shows altered heme binding compared to the wild-type enzyme
DELTA197-268
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mutation identified in female patient with microphthalmia with linear skin defects syndrome. In contrast to wild-type, mutant protein is unable to complement a Saccharomyces cerevisiae mutant deficient in the yeast enzyme ortholog. Upon expression in CHO-K1 cells, mutant protein fails to be sorted to mitochondria. Mutation results in disturbance of both oxidative phosphorylation and the balance between apoptosis and necrosis
R217C
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mutation identified in female patient with microphthalmia with linear skin defects syndrome. In contrast to wild-type, mutant protein is unable to complement a Saccharomyces cerevisiae mutant deficient in the yeast enzyme ortholog. Mutation results in disturbance of both oxidative phosphorylation and the balance between apoptosis and necrosis
additional information
E159A
site-directed mutagenesis of a domain II residue, the mutant shows altered heme binding compared to the wild-type enzyme
E159A
mutant displays enhanced release of cytochrome c from the active site. The mutant allows for synthesis of cytochrome c variants such as cyt c H19M (bis-Met), cyt c M81H (bis-His), cyt c M81A (His/OH?), cyt c C15S which exhibit proper folding
W118A
site-directed mutagenesis of a domain I residue, the mutant shows altered heme binding compared to the wild-type enzyme
W118A
mutant displays enhanced release of cytochrome c from the active site. The mutant allows for synthesis of cytochrome c variants such as cyt c H19M (bis-Met), cyt c M81H (bis-His), cyt c M81A (His/OH), cyt c C15S which exhibit proper folding
additional information
the mutants show altered heme binding with and/or without presence of cytochrome c, overview
additional information
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the mutants show altered heme binding with and/or without presence of cytochrome c, overview
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant GST-tagged enzyme and cytochrome c mutants from Escherichia coli strain RK103 membranes by ultracentrifugation, glutahione affinity chromatographyand ultrafiltration
recombinant GST-tagged wild-type and mutant enzymes and cytochrome c from Escherichia coli strain RK103 membranes by ultracentrifugation, glutahione affinity chromatographyand ultrafiltration
wild-type membrane-bound human GST-tagged enzyme with endogenous heme and in complex with its cognate human apocytochrome c and mutants from Escherichia coli
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
coexpression of N-terminally GST-tagged wild-type enzyme with endogenous heme and in complex with its cognate human apocytochrome c and mutants in Escherichia coli
recombinant coexpression of GST-tagged wild-type and mutant enzymes with cytochrome c in Escherichia coli strain RK103 membranes
recombinant coexpression of the GST-tagged enzyme with cytochrome c mutants, containing individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions of conserved motif CXXCH, in Escherichia coli strain RK103 membranes, single and double mutants form a complex with the enzyme but not the triple mutant
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
patient with deletion of the HCCS gene, diagnosis of a microphthalmia with linaer skin defects syndrome. Patient showed bilateral microphthalmia with optic atrophy, a congenital cataract, linear vascular lesions on the cheeks, neck and nose, apparently small, cupped poorly formed ears, anteverted nares, small areolae, a prominent xiphoid, an apparently small phallus, and right cryptorchidism. He had a patent ductus arteriosus, a patent foramen ovale and severe pulmonary hypertension attributed to pulmonary hypoplasia. Head ultrasound showed bilateral ventriculomegaly and agenesis of the corpus callosum. He also had poor tone and diminished reflexes on neurological examination. Patient died at 4 days of age
synthesis
mutants E159A and W118A display enhanced release of cytochrome c from the active site. The mutant allows for synthesis of cytochrome c variants such as cyt c H19M (bis-Met), cyt c M81H (bis-His), cyt c M81A (His/OH), cyt c C15S, which exhibit proper folding
medicine
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identification of mutations R217C and DELTA197-268 in female patients with microphthalmia with linear skin defects syndrome. In contrast to wild-type, mutant proteins are unable to complement a Saccharomyces cerevisiae mutant deficient in the yeast enzyme ortholog. Mutation results in disturbance of both oxidative phosphorylation and the balance between apoptosis and necrosis. Upon expression in CHO-K1 cells, mutant DELTA197-268 protein fails to be sorted to mitochondria
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Schaefer, L.; Ballabio, A.; Zoghbi, H.Y.
Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS)
Genomics
34
166-172
1996
Homo sapiens (P53701), Mus musculus (P53702)
Bernard, D.G.; Gabilly, S.T.; Dujardin, G.; Merchant, S.; Hamel, P.P.
Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases
J. Biol. Chem.
278
49732-49742
2003
Homo sapiens (P53701), Homo sapiens, Mus musculus (P53702), Saccharomyces cerevisiae
Wimplinger, I.; Morleo, M.; Rosenberger, G.; Iaconis, D.; Orth, U.; Meinecke, P.; Lerer, I.; Ballabio, A.; Gal, A.; Franco, B.; Kutsche, K.
Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome
Am. J. Hum. Genet.
79
878-889
2006
Homo sapiens
Qidwai, K.; Pearson, D.; Patel, G.; Pober, B.; Immken, L.; Cheung, S.; Scott, D.
Deletions of Xp provide evidence for the role of holocytochrome c-type synthase (HCCS) in congenital diaphragmatic hernia
Am. J. Med. Genet. A
152
1588-1590
2010
Homo sapiens (P53701)
Babbitt, S.E.; San Francisco, B.; Bretsnyder, E.C.; Kranz, R.G.
Conserved residues of the human mitochondrial holocytochrome C synthase mediate interactions with heme
Biochemistry
53
5261-5271
2014
Homo sapiens (P53701), Homo sapiens
Babbitt, S.E.; San Francisco, B.; Mendez, D.L.; Lukat-Rodgers, G.S.; Rodgers, K.R.; Bretsnyder, E.C.; Kranz, R.G.
Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, CysXXCysHis
J. Biol. Chem.
289
28795-18807
2014
Homo sapiens (P53701), Homo sapiens
San Francisco, B.; Bretsnyder, E.C.; Kranz, R.G.
Human mitochondrial holocytochrome c synthases heme binding, maturation determinants, and complex formation with cytochrome c
Proc. Natl. Acad. Sci. USA
110
E788-E797
2013
Homo sapiens (P53701), Homo sapiens
Babbitt, S.E.; Hsu, J.; Mendez, D.L.; Kranz, R.G.
Biosynthesis of single thioether c-type cytochromes provides insight into mechanisms intrinsic to holocytochrome c synthase (HCCS)
Biochemistry
56
3337-3346
2017
Homo sapiens (P53701)
Babbitt, S.E.; Hsu, J.; Kranz, R.G.
Molecular basis behind inability of mitochondrial holocytochrome c synthase to mature bacterial cytochromes Defining a critical role for cytochrome c alpha helix-1
J. Biol. Chem.
291
17523-17534
2016
Homo sapiens (P53701), Homo sapiens
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