1. Biochemistry and Chemical Biology

In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases

  1. Molly C Sutherland  Is a corresponding author
  2. Deanna L Mendez
  3. Shalon E Babbitt
  4. Dustin E Tillman
  5. Olga Melnikov
  6. Nathan L Tran
  7. Noah T Prizant
  8. Andrea L Collier
  9. Robert G Kranz  Is a corresponding author
  1. Department of Biology, Washington University in St. Louis, United States
  2. Department of Biological Sciences, University of Delaware, United States
https://doi.org/10.7554/eLife.64891
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Cite this article
  1. Molly C Sutherland
  2. Deanna L Mendez
  3. Shalon E Babbitt
  4. Dustin E Tillman
  5. Olga Melnikov
  6. Nathan L Tran
  7. Noah T Prizant
  8. Andrea L Collier
  9. Robert G Kranz
(2021)
In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases
eLife 10:e64891.
https://doi.org/10.7554/eLife.64891
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8 figures, 2 tables and 3 additional files

Figures

Figure 1 with 7 supplements
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Cyt c is biosynthesized in vitro by mitochondrial HCCS.

(a) Schematic of the in vitro heme attachment reaction of HCCS with apocytochrome c (apocyt c). Mitochondrial cyt c synthase, HCCS, positions heme (orange) and attaches it to apocyt c. Cyt c is …

Figure 1—figure supplement 1
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Cyt c attachment to heme and cyt c biogenesis pathways (Systems I, II, and III).

(a) Heme c synthesis (one vinyl group shown with one thiol of CXXCH): cys thiol attachment to α    carbon of the vinyl group of heme. (b) schematic of the reaction that takes place between apocyt c …

Figure 1—figure supplement 2
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Titration of heme loading (HL) in GST-HCCS.

WT (blue, n = 7) and H154A (orange, n = 3). Exogenous heme at indicated concentration (x-axis) was added to GST-HCCS during batch purification with glutathione agarose for 20 hr. GST-HCCS was …

Figure 1—figure supplement 3
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HCCS in vitro biosynthesized cyt c has heme attached and is properly folded.

(a) The reaction of 10 µM HCCS and 20 µM apocyt c (initial, black) was initiated with 5 mM DTT and incubated for 1 hr at 37°C (red, final). The inset shows the pyridine hemochrome assay spectra …

Figure 1—figure supplement 4
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In-vitro biosynthesis of cyt c by HCCS is temperature dependent.

Aerobic reaction with 3 µM HCCS (+HL) and 20 µM apocyt c were initiated with 3 mM DTT and monitored over a period of 4 hr at the indicated time points and temperatures. Cyt c biosynthesis was …

Figure 1—figure supplement 5
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HCCS function is dependent on the presence of DTT (aerobic conditions).

(a) 0 mM, (b) 0.1 mM, (c) 1 mM, and (d) 3 mM DTT were added to a reaction of 3 µM HCCS and 6.5 µM apocyt c. Cyt c biosynthesis was monitored by appearance of a 550 nm peak. The initial (black) …

Figure 1—figure supplement 6
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Aerobic in vitro reaction with HCCS + apocyt c.

(a) As in Figure 1b, apocyt c and HCCS (± HL) were combined (black). The reaction was initiated with 5 mM DTT and allowed to react for 1 hr at 37°C (red). HCCS matured apocyt c, while HCCS H154A did …

Figure 1—figure supplement 7
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Model for HCCS function proposed previously based on in vivo results (Babbitt et al., 2015; San Francisco et al., 2013).

(a) Four-step model of HCCS function. UV–vis spectra corresponding to each step are shown. Step 1, HCCS binds to heme via residue H154. Step 2, HCCS binds to apocyt c and His19 (of CXXCH) forms the …

Figure 2
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Cyt c biosynthesized in vitro is released by mitochondrial HCCS.

(a) Schematic of HCCS released product assay. In vitro reaction is carried out with bead tethered GST-HCCS. Centrifugation separates the beads (GST-HCCS) and supernatant (e.g. released products). (b)…

Figure 3 with 3 supplements
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HCCS requires alpha helix 1 of cyt c for heme attachment to peptides containing CXXCH.

(a) Sequence of three CXXCH containing peptides with alpha helix 1 and CXXCH designated. Three-dimensional structures of peptides with heme were generated from the cyt c 3D crystal structure PDB: …

Figure 3—figure supplement 1
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Attachment of heme to peptides by HCCS.

(a) In vitro assay was performed with HCCS with the labeled peptides. The final UV–vis heme spectra is shown. A peak at 550–555 nm indicates that heme has covalently bound to cyt c peptide. Peaks at …

Figure 3—figure supplement 1—source data 1

ImageJ Pixel analysis of heme stained bands.

https://cdn.elifesciences.org/articles/64891/elife-64891-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
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HCCS anaerobic in vitro attachment of heme to CXXCH variant peptides.

C15S refers to Cys15Ser; DC15 refers to Cys15 D-Cys15 replacing L-Cys15; HoC15 refers to homocysteine replacing L-Cys15. (a) Heme stain and Coomassie showing that 20mer peptides WT (lane 2), C15S …

Figure 3—figure supplement 3
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In vitro HCCS attachment of heme to C15S 20mer and analysis of release.

(a) Release assay as in Figure 4b. Briefly, C15S 20mer is added to HCCS bound to glutathione agarose (beads) and reacted for 1 hr. Heme staining shows that the majority of matured C15S 20mer remains …

Figure 3—figure supplement 3—source data 1

Summary of percent released substrate.

https://cdn.elifesciences.org/articles/64891/elife-64891-fig3-figsupp3-data1-v1.xlsx
Figure 4
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Peptides not released by HCCS can inhibit HCCS in vitro biosynthesis of cyt c.

(a) The 20mer reaction was analyzed by SEC-HPLC (blue) and compared to HCCS alone (brown). The ‘heme attached peptide’ serves as a positive control for a released peptide (green). It is commercially …

Figure 5
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Construction of CcsBA with a C-terminal 6XHis affinity tag.

(a) Schematics of CcsBA constructs used for overexpression and affinity purification. Gray, GST ORF; red, ccsB; blue ccsA; purple, C-terminal 6XHis tag. Site of natural proteolysis is shown with …

Figure 6
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In vitro biosynthesis of cyt c by the bacterial synthase, CcsBA.

(a) Schematic of the heme attachment reaction of CcsBA with apocyt c. Note, CcsBA has two heme binding sites, one in the periplasmic WWD domain (P-heme site, orange heme) and one in the …

Figure 7 with 3 supplements
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CcsBA recognition of peptides containing CXXCH for heme attachment.

Peptides are described in Figure 3a. (a) Ten micromolar of CcsBA was incubated with 20 µM of the indicated peptide for 3 hr. Samples were taken at 0 and 3 hr and separated by Tris–tricine SDS–PAGE. …

Figure 7—figure supplement 1
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Attachment of heme to peptides by CcsBA.

(a) In vitro assay was performed with CcsBA with the indicated peptides. The final UV–vis heme spectra is shown. A peak at 550–555 nm indicates that heme has covalently bound to cyt c peptide. Peaks …

Figure 7—figure supplement 1—source data 1

Image J Pixel analysis of heme-stained bands.

https://cdn.elifesciences.org/articles/64891/elife-64891-fig7-figsupp1-data1-v1.xlsx
Figure 7—figure supplement 2
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CcsBA and HCCS in vitro heme attachment to peptides.

(a) Heme and coomassie stain of anaerobic in vitro reactions of CcsBA and HCCS showing that CcsBA matures the 20, 16, and 11mers, while HCCS matures the 16 and 20mers under the same conditions at 3 …

Figure 7—figure supplement 3
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CcsBA anaerobic in vitro attachment of heme to CXXCH peptide variants.

C15S refers to Cys15Ser; DC15 refers to Cys15 d-Cys15 replacing l-Cys15; HoC15 refers to homocysteine replacing l-Cys15. (a) Heme stain of CcsBA and CXXCH variant 20mer peptides (red) showing that …

Figure 8
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Modeled structure of HCCS using GREMLIN/Rosetta approach.

(a) Electrostatic view of HCCS structure. Acidic surfaces are red; basic surfaces are blue. Heme is modeled with H154 ligand, within a pocket surrounded by acidic residues. Ribbon diagram of HCCS …

Tables

Table 1
Attachment of heme to peptides* by HCCS and CcsBA.
                                                   HCCS CcsBA
Peptide name Peptide sequence Attachment α-Peak (nm) # cov
attachments**		 Attachment α-Peak (nm) # cov
attachments**
Minimal recognition primary/secondary sequences (lengths)
HH Cyt c …GDVEKGKKIFVQK CAQCHTVE… Attached 550 2 Attached 550, 560 2
56-mer GDVEKGKKIFIMK CSQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGYSYTAANKNKG Attached 555 2 Attached 550, 560 2
20 mer GDVEKGKKIFIMK CSQCHTV Attached 553–554 2*** Attached 550, 560 2
16 mer KGKKIFIMK CSQCHTV Attached 552 2 Attached 551, 560 2
11 mer IMK CSQCHTVE Not attached 559 0 Attached 550, 560 2
nine mer K CSQCHTVE Not attached 559 0 Attached 550, 560 2
Cysteine substitutions
20 mer Cys15S GDVEKGKKIFIMK SSQCHTV Attached 555,559.5 1*** Not attached n.a. 0
20 mer DCys15 GDVEKGKKIFIMK(D-C)SQCHTV Attached 555 2*** Not attached 560 0
20 mer HoCys15 GDVEKGKKIFIMK HoCSQCHTV Attached 555 1*** Attached 550, 560 2
20 mer Cys18S GDVEKGKKIFIMK CSQSHTV Attached 559 1*** Not attached 560 0
20 mer DCys18 GDVEKGKKIFIMK CSQ(D-C)HTV Attached 554 1*** Not attached 560 0
20 mer HoCys18 GDVEKGKKIFIMK CSQHoCHTV Attached 558 1*** Not attached 560 0
20 mer Cys15S/Cys18S GDVEKGKKIFIMK SSQSHTV Not attached 559 0 Not attached 560 0
Histidine (of CXXCH) and lysine (K→D) substitutions for testing interaction models
20mer H19A GDVEKGKKIFIMK CSQCATV Not attached 560 0 Not attached 560 0
20mer H19M GDVEKGKKIFIMK CSQCMTV Attached 559 2 Not attached 560 0
20mer H19K GDVEKGKKIFIMK CSQCKTV Not attached 560 0 Not attached 560 0
20mer K6A, K8D, K9D, K14D GDVEAGDDIFIMD CSQCHTV Not attached 560 0 Not attached 560 0

  1. *Supplementary file 2 contains additional information about peptides, eg. purity, synthesis co, and presence or absence of an N-terminal biotin-AHX tag.

    **Number of covalent attachments determined by the final reaction spectra absorbance blue shifted from 560 nm and the presence of a heme stainable peptide.

  2. ***Pyridine hemochrome was performed to determine this number.

Key resources table
Reagent type (species) or
resource		 Designation Source or reference Identifiers Additional information
Strain, strain background (Escherichia coli) NEB 5-α New England Biolabs fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 Electrocompetent cells
Strain, strain background (E. coli) C43(DE3) doi:10.1006/jmbi.1996.0399; Miroux and Walker, 1996 F – ompT hsdSB (rB- mB-) gal dcm (DE3) Electrocompetent cells
Strain, strain background (E. coli) RK103 doi:10.1111/j.1365–2958.2006.05132.x MG1655 Δccm::kanR , deleted for all ccm genes Electrocompetent cells, protein expression, functional assays
Strain, strain background (E. coli) MS36 doi:10.1128/mBio.02134–18 C43 Δccm::kanR , deleted for all ccm genes Electrocompetent cells, protein expression, functional assays
Antibody Anti-equine heart cytochromec (Rabbit polyclonal) doi:10.1074/jbc.M116.741231 (1:10,000)
Recombinant DNA reagent pRGK332 (plasmid) doi:10.1111/j.1365–2958.2006.05132.x pBAD Bordetella pertussis cytochrome c4:His
Recombinant DNA reagent pRGK368 (plasmid) doi:10.1128/JB.01388–06; Richard-Fogal et al., 2007 pGEX Helicobacter hepaticus GST:CcsBA
Recombinant DNA reagent pRGK403 (plasmid) doi:10.1073/pnas.1213897109 pGEX GST:HCCS
Recombinant DNA reagent pRGK420 (plasmid) doi:10.1073/pnas.1213897109 pGEX GST:HCCS H154A
Recombinant DNA reagent pMCS97 (plasmid) This study pGEX H. hepaticus GST:CcsBA:His See Materials and Methods and Supplementary file 1
Recombinant DNA reagent pMCS64 (plasmid) This study pGEX H. hepaticus GST*CcsBA:His See Materials and Methods and Supplementary file 1
Recombinant DNA reagent pMCS154 (plasmid) doi:10.1128/mBio.02134–18 pGEX H. hepaticus GST:CcsBA:His
Recombinant DNA reagent pMCS558 (plasmid) This study pGEX H. hepaticus *CcsBA:His See Materials and Methods and Supplementary file 1
Recombinant DNA reagent MCS598 (plasmid) This study pGEX H. hepaticus GST*CcsBA:His P-His1/2G See Materials and Methods and Supplementary file 1
Sequence-based reagent pGEX GST*F This study PCR Primer tcggatctggttccgcgttgaaggaggaaggatccatgatgaat
Sequence-based reagent pGEX GST*R This study PCR Primer attcatcatggatccttcctccttcaacgcggaaccagatccga
Sequence-based reagent pGEX CcsBA 6HisF This study PCR Primer gagtgcttgatatgccccatttacatcaccatcaccatcactaactcgagcggc
Sequence-based reagent pGEX CcsBA 6HisR This study PCR Primer gccgctcgagttagtgatggtgatggtgatgtaaatggggcatatcaagcactc
Sequence-based reagent MSP5 This study PCR Primer gtgcttaaatcttattggctcaacattggcgtctccgtcatca
Sequence-based reagent MSP6 This study PCR Primer tgatgacggagacgccaatgttgagccaataagatttaagcac
Sequence-based reagent MSP7 This study PCR Primer ttattatctcacaggtatgggcagctatgccgcaggagaa
Sequence-based reagent MSP8 This study PCR Primer ttctcctgcggcatagctgcccatacctgtgagataataa
Peptide, recombinant protein Holo-MP11 Sigma-Aldrich Cat. #M6756 See Supplementary file 2
Peptide, recombinant protein Biotin-56-mer RS-synthesis See Supplementary file 2
Peptide, recombinant protein Biotin-20 mer RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20-mer RS-synthesis See Supplementary file 2
Peptide, recombinant protein Biotin-16 mer RS-synthesis See Supplementary file 2
Peptide, recombinant protein 11 mer RS-synthesis See Supplementary file 2
Peptide, recombinant protein nine mer-biotin RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20 mer Cys15S RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20 mer DCys15 CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20 mer HoCys15 CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20 mer Cys18S CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20 mer DCys18 CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20 mer HoCys18 CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20 mer Cys15S/Cys18S CS Bio Co See Supplementary file 2
Peptide, recombinant protein 20mer H19A RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20mer H19M RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20mer H19K RS-synthesis See Supplementary file 2
Peptide, recombinant protein 20mer K6A, K8D, K9D, K14D RS-synthesis See Supplementary file 2
Peptide, recombinant protein Pierce-glutathione agarose Thermo Scientific Cat. #16101
Peptide, recombinant protein Talon Resin TaKaRa Cat. #635503
Chemical compound, drug Hematin Fisher Cat. #AAA1851803
Chemical compound, drug N, N, N’, N’-tetramethylbenzidine (TMBZ) Sigma Cat. #1086220001
Chemical compound, drug Equine horse-heart cytochrome c Sigma Cat. #C2506
Chemical compound, drug 2,6-dichloroindophenolate hydrate (DCPIP) Sigma Cat. #D-1878
Commercial assay or kit Pierce-SuperSignal West Femto ECL reagent Thermo Scientific Cat. #PI34096

Additional files

Supplementary file 1

Relevant strains, plasmids, and primers.

https://cdn.elifesciences.org/articles/64891/elife-64891-supp1-v1.docx
Supplementary file 2

Apo peptides used for in vitro assays.

https://cdn.elifesciences.org/articles/64891/elife-64891-supp2-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/64891/elife-64891-transrepform-v1.docx

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