Using purified human HCCS, we reconstituted cyt c synthase activity with equine apocyt c as substrate, initially assaying formation of a peak at 550 nm, diagnostic of cyt c’s typical UV–vis spectra (Figure 1a). Recombinant human HCCS (GST-tagged) is functional in vivo, attaching heme to co-expressed apocyt c (San Francisco et al., 2013) in E. coli. We have previously shown that HCCS co-purifies with heme, which is liganded to His154 (San Francisco et al., 2013). UV–vis spectra of purified HCCS shows a 423 nm and broad 560 nm absorption, typical of heme proteins, while HCCS H154A variant does not bind heme (Figure 1b, −HL). We developed a ‘heme-loading (HL)’ protocol to increase the levels of heme bound in HCCS (+HL, ~30% occupied) above the co-purified levels of endogenous heme (−HL, ~10% occupied). HL was also advantageous since the HL protocol removes excess heme, thus minimizing spectral interference from free heme in reactions. HL was shown to depend on the natural His154 ligand (Figure 1b, +HL black line), and loading was saturated at 2–5 µM heme (Figure 1—figure supplement 2). Initial reconstitutions were performed with wild type (wt) HCCS (±HL) and the HCCS His154Ala variant that does not bind heme (Figure 1b). Upon incubation for 1 hr in the presence of apocyt c and dithiothreitol (DTT), a sharp 550 nm peak emerged, indicative of a c-type cytochrome (Figure 1b, red with wt). This occurred with wt HCCS containing endogenous heme (−HL) and in vitro loaded heme (+HL), while HCCS H154A did not produce the 550 nm peak. A second method to determine if heme has been covalently attached to the apocyt c is to separate reactions with denaturing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by heme staining, whereby covalently attached heme electrophoreses with the polypeptide (Figure 1c). Reactions with wt HCCS (−HL and +HL) and apocyt c confirmed that heme is covalently attached to cyt c (12 kDa) in the 1 hr reaction (Figure 1c, lanes 3, 8). As expected, no cyt c was formed with the HCCS H154A variant (Figure 1c, lanes 5, 10). Pyridine hemochrome spectra is often used to determine if two, one, or no covalent bonds to heme are present, with two thioether bonds showing a 550 nm peak (c-heme) and 560 nm for none (b-heme). The in vitro synthesized product has two thioether bonds, indicated by a 550 nm peak in pyridine hemochrome spectra (Figure 1—figure supplement 3a).

In vitro reconstitutions were studied for optimal conditions and requirements. Synthesis is optimal at 37°C (Figure 1—figure supplement 4), required DTT (Figure 1—figure supplement 5), with the cyt c product observed in 10 min (e.g. Figure 1d, lane 5). While cyt c is formed in both aerobic (Figure 1—figure supplement 6) and anaerobic conditions (Figure 1b,c), we decided to use anaerobic conditions for all studies since peptide substrates (below) under aerobic conditions required varying DTT concentrations, likely due to distinct thiol reducing requirements of individual peptides in air.

To further characterize HCCS, substrates and products, we employed analytical HPLC size exclusion chromatography (SEC), whereby UV–vis spectra of each separated species was recorded (Figure 1e). HCCS (brown profile) elutes earlier than cyt c (green profile), and because these are 424 nm (heme) profiles, it is observed in the reaction (blue profile) that heme in HCCS decreases while cyt c product increases. These results also demonstrate that the cyt c product is released from the HCCS active site since it elutes at the same time as purified cyt c (holocyt c). We conclude that we have recapitulated in vitro the four-step process proposed previously (Figure 1—figure supplement 7) for HCCS-mediated cyt c biogenesis: heme binding (step 1), apocyt c binding (step 2), thioether formation (step 3), and release (step 4) (Babbitt et al., 2015; San Francisco et al., 2013). Next, we further characterize the released cyt c product to establish whether proper folding to the native state resulted from in vitro biogenesis.

We developed a HCCS-tethered (to glutathione beads) release assay to isolate HCCS reaction product(s), confirm that cyt c is released, and obtain high yields for product characterization (Figure 2a). Spectra of the released product (Figure 2b) is identical to holocyt c. SDS–PAGE of stages in the bead release protocol (Figure 2c) showed a released product of 12 kD that heme stained and reacted with cyt c antisera (Figure 2c, lane 2). We determined spectrally that the released cyt c has folded properly, forming the Met81 ligand as well as His19 (Figure 1—figure supplement 3b). Redox titrations (Figure 2d) showed that the redox potential of the cyt c in vitro product is the same as cyt c produced in vivo, +253 mV (Figure 2d). Analyses of supernatants (released), washes, and bead-retained material allowed for an estimate that at least 62% of cyt c is released from HCCS (Figure 2c). Since heme in all cyt c’s is attached stereochemically (Figure 2e), we performed circular dichroism (CD) spectra to compare the released (in vitro) product to cyt c made in vivo (Figure 2f). CD absorption of heme (~420 nm) is reduced in globins when heme binds in multiple orientations compared to a single orientation (Aojula et al., 1986; Nagai et al., 2014). Cyt c synthesized in vitro by HCCS shows an identical CD spectral profile as in vivo synthesized (Figure 2f). We conclude that in vitro reconstitution with purified HCCS results in stereochemical heme attachment, release, and proper folding of cyt c.

In vitro reconstitution of the cyt c synthases provides an opportunity to investigate chemically synthesized apocyt c peptides and analogs as substrates. For example, there are in vivo genetic results suggesting that alpha helix 1, adjacent to the CXXCH motif (Figure 3a), of native cyt c is necessary for maturation by HCCS (San Francisco et al., 2013; Zhang et al., 2014; Kleingardner and Bren, 2011). In fact, the bacterial cyt c has a natural deletion of Met13 in alpha helix 1, recently shown in vivo to be the basis for the inability of HCCS to mature bacterial cyt c (Babbitt et al., 2016; Verissimo et al., 2012). We wanted to determine if cyt c peptides are recognized in vitro and if so the minimal sequence for recognition and heme attachment. Initially, we examined three peptides, an 11mer, 16mer, and 20mer with the 11mer lacking the sequence of alpha helix 1 (Figure 3a). Heme stains of tricine SDS–PAGE were used to detect whether heme was covalently attached to peptides (Figure 3b). After 1 hr, reactions showed that the 16mer (Figure 3b, lane 6) and 20mer (Figure 3b, lane 8) possessed an intense heme-stained peptide of 2.8 kDa, whereas the 11mer did not (Figure 3b, lane 4). Spectral analyses showed that the 11mer reaction looked like HCCS alone (no peptide added), whereas the reactions with the 16mer and 20mer showed a 552–553 nm peak (Figure 3c). We have previously shown that some recombinant HCCS is co-purified with cyt c remaining bound (and heme attached) (San Francisco et al., 2013). UV/vis absorption of these HCCS/cyt c complexes exhibits a peak in the reduced state of 553–555 nm (Figure 1—figure supplement 7), whereas a purified heme attached peptide shows a 550 nm peak (Figure 1—figure supplement 7b). Spectral results of HCCS reactions with 16mer and 20mer peptides (i.e. 552–553 nm peaks, see Figure 3c) suggest heme is covalently attached to the peptides, but that they remain in complex with HCCS, unlike full-length cyt c produced in vitro. To further test CXXCH peptide recognition, we tested a 56mer (with alpha helix 1 and 2 of cyt c) and a 9mer (Figure 3—figure supplement 1). While the 56mer was recognized and heme attached, the 9mer was not, consistent with the in vivo results that alpha helix 1 is required for heme attachment (Babbitt et al., 2016). Because HCCS reaction with the 56mer yields a 555 nm absorption (Figure 3—figure supplement 1a), it is likely not released.

To confirm that heme-attached peptides remain bound to HCCS, we used both HPLC SEC and the bead release assay described above (Figure 2a). HPLC separation (Figure 4a) showed that HCCS with the 20mer reaction (blue profile) eluted at the same time as HCCS alone (brown profile), not unexpected since a small 2.8 kD unreleased product would not significantly alter size exclusion properties. However, the spectra of the 20mer reaction from the HPLC SEC shows the signature of a HCCS-bound cyt c product, with a peak at 553 nm. This supports the conclusion that the heme attached 20mer remains bound to HCCS upon HPLC SEC, explaining why no heme-peptide product elutes separately (Figure 4a, compare blue and green profiles). Results of the bead release assay also show there is very little release of the heme-attached peptides from HCCS. Spectra of the reaction supernatant (red) exhibits very little heme (Figure 4b), unlike with full cyt c (Figure 2b, red). However, eluted HCCS from the beads show a spectra consistent with heme-attached peptide still bound, with a 555 nm peak (Figure 4b, purple). Quantitation of the level of heme-attached 20mer released from HCCS was carried out using the bead release assay (Figure 4c), with 14 ± 3% of the heme-attached peptide released from HCCS.

We evaluated whether peptides recognized by HCCS would act as inhibitors of heme attachment to subsequent addition of apocyt c. We carried out reactions with the three peptides for 1 hr, then added apocyt c, taking samples throughout (Figure 4d). The 11mer behaved as expected, as if no other substrate was present, with synthesis of cyt c occurring (in Figure 4e, compare lanes 1–4 and 5–8 boxed bands). This also suggests that the 11mer is not recognized by HCCS, in that it does not prevent apocyt c from binding. However, both the 16mer (Figure 4e, lanes 9–12) and 20mer (lanes 13–16) showed heme attached to the peptides, but not to the apocyt c. We consider this inhibition of cyt c biogenesis (see Discussion). We conclude that alpha helix 1 is necessary and sufficient for recognition and attachment to the adjacent CXXCH motif. Our findings suggest that folding of cyt c is required for optimal release from the HCCS active site (see Discussion).

Our previous studies with CcsBA have used recombinant GST-tagged CcsBA (from Helicobacter), shown to be functional in vivo and co-purify with endogenous heme (Feissner et al., 2006; Frawley and Kranz, 2009; Sutherland et al., 2018b). We concluded that CcsBA is both a heme exporter and a cyt c synthase with two heme binding sites (Figure 6a). To increase CcsBA yields for in vitro and future structural studies, we explored various tagging and expression strategies, ultimately selecting a C-terminal hexahistidine tagged CcsBA which gave high yields (Figure 5a). For unknown reasons, yields were higher when the GST ORF (with stop codon), as well as a new ribosome binding site upstream of ccsBA were used (threefold higher than GST-tagged or without the GST gene: Figure 5a,b). The purified hexahistidine tagged CcsBA still possessed the natural proteolysis site we have previously characterized (Frawley and Kranz, 2009; Sutherland et al., 2018b), resulting in two polypeptides (Figure 5c, lane 4, boxed). The GST*CcsBA:His construct is hereafter referred to as CcsBA:His. Using the anaerobic in vitro reconstitution conditions described above for HCCS, both the purified GST-CcsBA and metal-affinity purified CcsBA:His, both with endogenous heme, were active for heme attachment to apocyt c in vitro (Figure 6a–d). For further studies here, we used the CcsBA:His due to its higher yields. We have previously shown that while wt CcsBA has heme in both the P-His/WWD and TM-His sites (Figure 6a), the P-His variants possess heme only in the TM-His site (Sutherland et al., 2018b). GST:CcsBA P-His mutants are unable to attach heme in vivo to cyt c4, yet co-purified with heme (Sutherland et al., 2018b). Since heme is proposed to attach to apocyt c from the P-His/WWD site (Figure 6a), we tested whether the P-His variant functions in vitro, representing ideal negative controls for genuine in vitro attachments. Importantly, the GST:CcsBA P-His variant did not attach heme to apocyt c in vitro (Figure 6b). In vitro reactions with the wt CcsBA:His shows initial spectral signatures of b-heme (Figure 6c, black spectra). Within 1–3 hr, the wt CcsBA shows two peaks of reduced heme, one at 560 nm and a 550 nm peak that is characteristic of covalent heme attached in c-type cytochromes (Figure 6c, red spectra). It is likely that the b-heme (in the TM-His site) is responsible for the absorption remaining at 560 nm. These results were confirmed by SDS–PAGE and heme stains at the different time points (Figure 6d), confirming that the wt CcsBA formed cyt c. We conclude that purified wt CcsBA acts as a cyt c synthase in vitro and that heme is attached from the P-His/WWD domain, as hypothesized from in vivo results.

A time course of in vitro reactions with wt CcsBA shows that the covalent attachment to apocyt c is measurable at 20 min, reaching a maximum at approximately 3 hr (Figure 6e). Spectra at selected time points confirm these results (Figure 6f, see 550 nm formation). To determine whether cyt c is released from CcsBA and folds into its native state, we performed HPLC SEC on CcsBA alone and from a 3 hr reaction with apocyt c (Figure 6g). CcsBA in vitro synthesized cyt c is released and elutes at the same position as purified cyt c. The cyt c product (Figure 6g, last inset) is spectrally identical to cyt c produced by HCCS in vitro and to purified cyt c generated in vivo. We conclude that apocyt c is matured and released by CcsBA in vitro, with folding of cyt c into its native state.

Similar to HCCS studies, we used the 11, 16, and 20mer peptides (Figure 3a) and heme staining of tricine SDS–PAGE, to determine whether CcsBA attaches heme to peptide substrates and if so, what sequence or structural requirements are important. In CcsBA in vitro reactions, the 20mer, 16mer, and 11mer peptides each resulted in covalent heme after 3 hr in vitro reactions (Figure 7a). Spectral analyses also showed formation of 550 nm peaks (Figure 7b), including reactions with the 11mer, which was not recognized by HCCS. Because the 560 nm peak also remains in reactions, likely due to heme in the TM-His site, we used second-derivative spectra to delineate and quantitate the levels of attached heme present (Figure 7b, last panel, 550 nm), also confirming that all peptides possess the 550 nm absorption characteristic of c-type heme. The 56mer (alpha helix 1 and 2 of cyt c) and 9mer were also recognized and attached to heme by CcsBA (Figure 7—figure supplement 1). We conclude that the bacterial CcsBA cyt c synthase does not require the alpha helix 1 and that the recognition requirements are different than the mitochondrial HCCS (see Figure 7—figure supplement 2 for parallel reactions of HCCS and CcsBA.)

The ability to biosynthesize heme-attached CXXCH peptides in vitro by HCCS and CcsBA facilitated a more detailed analysis of the cysteines and histidine in the substrates. For example, cysteine substitutions in the chemically synthesized peptides would determine whether each cysteine is required and whether non-standard thiol amino acids are recognized (Table 1). Homocysteine (HoC) has an additional carbon between the thiol and alpha carbon, while d-cysteine (D-C) rotates the thiol sidechain (see Table 1 for structures). All substitutions were synthesized in the 20mer background since both HCCS and CcsBA could attach heme to it and the product is easily detected on heme stains of tricine SDS–PAGE. That is, if the peptide product has a covalent attachment, it will migrate at 2.8 kD and stain for heme (Figure 3b; Figure 7a; Figure 7—figure supplement 2). This method does not indicate whether a modified thiol (HoC or D-C) has a covalent attachment, so we also performed UV–vis and pyridine hemochrome spectroscopy to provide evidence of thioether formation. Figure 3—figure supplement 2 and Figure 7—figure supplement 3 show results of in vitro reactions of HCCS and CcsBA with the peptide analogs, as summarized in Table 1.

In the case of HCCS, all 20mer peptide variants possessed at least one covalent attachment with the exception of the SXXSH variant (Table 1, blue highlights). This indicates that HCCS does not require both cysteines for recognition, a conclusion consistent with in vivo results of engineered cyt c substrate variants (Babbitt et al., 2014). Importantly, the HCCS/peptide complexes exhibit spectral signatures of purified HCCS/cyt c co-complex variants produced in vivo (Babbitt et al., 2017). For example, HCCS reactions with the SXXCH peptide shows a split alpha peak at 555/560 nm (Figure 3—figure supplement 2b), just as shown in vivo with the HCCS/Cys15Ser variant (Babbitt et al., 2017). Pyridine hemochrome spectra of HCCS reaction products were used to show whether the non-natural thiols were covalently attached. Both homocysteine and the DCys18 (Figure 3—figure supplement 2f,g), thiols were not attached, possessing only a single thioether, resulting in a hemochrome spectral peak of 552 nm that reflected attachment to Cys15. However, the DCys15 variant possessed two thioether attachments, thus both thiols reacted (Figure 3—figure supplements 2c, 550 nm pyridine hemochrome peak). This indicates that rotation of the first thiol (Cys 15) of the CXXCH substrate is more permissive at the active site of HCCS.

Lastly, we examined the role of the conserved H19 of the CXXCH motif. 20mer peptide were synthesized with H19M, H19A, and H19K substitutions. The H19A and H19K variants did not attach heme, while the H19M variant attached heme at low levels (Figure 3—figure supplement 1), suggesting methionine can act as a weak ligand in HCCS.

In the case of the bacterial CcsBA, an entirely different set of rules emerge for CXXCH substrate recognition (Table 1, compare blue to orange highlighted variants). Only one 20mer cysteine variant showed any covalent attachment: the first cysteine thiol replaced with a homocysteine (Figure 7—figure supplement 3f). The HoCys15 variant has two covalent linkages (550 nm peak), suggesting that the first thiol is more permissive in distance from the alpha carbon (i.e. of the first cysteine of CXXCH). Because DCys15 was not attached, unlike with HCCS, rotation of the first thiol may be less permissive at the CcsBA active site. No 20mers with histidine substitutions possess covalently attached heme with CcsBA (Figure 7—figure supplement 1).

Escherichia coli strains were grown in Luria-Bertani (LB; Difco) broth with selective antibiotics and inducing reagents as required. Antibiotic/induction concentrations: carbenicillin, 50 µg/ml; chloramphenicol, 20 µg/ml, isopropyl β -D-1-thiogalactopyranoside (IPTG; Gold Biotechnology), 1.0 mM or 0.1 mM; arabinose (alfa Aesar), 0.2% (wt/vol).

Cloning was performed using E. coli NEB-5  α with the QuikChange II site-directed mutagenesis kit (Agilent Technologies) following the manufacturer’s instructions. Strains, plasmid, and primer lists are provided in Supplementary file 1 in supplemental material.

GST-HCCS purifications were performed as previously described (San Francisco et al., 2013). Briefly, starter cultures (100 ml) were grown overnight at 37°C and 200 rpm. Starter cultures were used to inoculate 1 l of LB supplemented with appropriate antibiotics. One liter cultures were grown at 37°C and 120 rpm for 1 hr, and next expression of GST-HCCS was induced with 0.1 mM IPTG. Cells were harvested after 5 hr by centrifugation at 4500 g and cell pellets were stored at −80°C. Cell pellets were resuspended in PBS supplemented with 1 mM phenylmethansulfonul fluoride (PMSF), lysed by sonication (Branson250 sonicator), and cleared of cell debris by centrifugation at 24,000 g for 30 min at 4°C. Separation of soluble and membrane fractions was achieved by high-speed ultracentrifugation at 100,000 g for 45 min at 4°C. Membrane pellets were solubilized in 50 mM Tris pH 8, 150 mM NaCl, and 1% Triton X-100 for 1 hr on ice. Solubilized membranes were added to glutathione agarose (Pierce) for batch pulldown. Note, GST-HCCS used for in vitro reactions were heme loaded at this step by addition of 5 µM hemin during batch pulldown (see below). Columns were washed by gravity flow and eluted in 50 mM Tris pH8, 150 mM NaCl, and 0.02% Triton X-100 supplemented with 20 mM glutathione. Elution was concentrated using Amicon Ultra Centrifugal Filters (Millipore), and protein concentration was determined by Bradford assay (Sigma).

GST-CcsBA and GST-CcsBA:His purifications were performed as previously described (Frawley and Kranz, 2009; Sutherland et al., 2018b). Briefly, 5 ml starter cultures were grown for ~8 hr at 37°C with rocking. Starter cultures were diluted 1:200 into 1 l LB with selective antibiotics and grown overnight at 24°C and 240 rpm to saturation. Expression of GST-CcsBA was induced with 1 mM IPTG for 6 hr, cells were harvested at 4500 g, and cell pellets were stored at −80°C. Cell pellets were resuspended in Resin Buffer (20 mM Tris pH8, 100 mM NaCl) supplemented with 1 mM PMSF and 1 mg/ml egg white lysozyme (Sigma-Aldrich). Cells were lysed, cleared of debris, and separation of membrane fraction was performed as described for GST-HCCS above. Membrane pellets were solubilized in Resin Buffer with 1% n-dodecyl-β-d-maltopyranoside (DDM; Anatrace) and batch purified for 2 hr with glutathione agarose (Pierce). Columns were washed by gravity flow using Resin Buffer with 0.02% DDM and eluted in Resin Buffer with 0.02% DDM and 20 mM glutathione. Elution was concentrated using Amicon Ultra Centrifugal Filters (Millipore), and protein concentration was determined by Bradford assay (Sigma).

GST-CcsBA:His, GST*CcsBA:His and *CcsBA:His were performed as described above for GST-CcsBA with the following modifications. Batch pulldowns were performed using Talon Affinity Metal Resin (Takara). Gravity flow washes were performed in Resin buffer with 0.02% DDM supplemented with 0 mM imidazole (wash 1), 2 mM imidazole (wash 2), and 5 mM imidazole (wash 3). Protein was eluted in Resin Buffer with 0.02% DDM and 125 mM imidazole. Elution was concentrated using Amicon Ultra Centrifugal Filters (Millipore), and protein concentration was determined by Bradford assay (Sigma).

To increase heme co-purification of GST-HCCS, exogenous heme was added to the affinity purification, resulting in ‘heme loaded’ HCCS. During binding of the solubilized membrane preparations to glutathione agarose, hemin (1.3 mg/ml in DMSO) was added to a final concentration of 5 µM. Heme loading increases HCCS heme co-purification from ~10% to ~30%. To determine the optimal concentration of hemin, a range of values was tested (see Figure 1—figure supplement 2). After batch affinity purification, the column was washed (removing unbound hemin) and eluted as described in protein purification section.

Samples were prepared in loading dye at 1:1 (v/v) that did not contain reducing agents and were not boiled to maintain heme signals. Samples were separated by SDS–PAGE or Tricine SDS–PAGE (peptides). Heme staining was performed by transfer to nitrocellulose and detection of heme signal using the SuperSignal Femto kit (Pierce) (Feissner et al., 2003), with imaging on a LI-COR odyssey Fc (LI-Cor Biosciences) or by in-gel heme stain with N, N, N’, N’-tetramethylbenzidine (TMBZ) (Feissner et al., 2003; Francis and Becker, 1984; Thomas et al., 1976). Total protein was detected by staining SDS–PAGE gels with Coomassie stain or nitrocellulose blots with SYPRO Ruby Blot Stain according to the manufacturer’s instructions (Molecular Probes). Immunoblots using an antibody specific to equine heart cytochrome c (Cocalico Biologics) were performed as previously described (Babbitt et al., 2016).

UV–vis absorption spectroscopy was obtained with a Shimadzu UV-1800 spectrophotometer. Spectra were recorded in the assay buffer and under aerobic or anaerobic conditions as indicated. Heme quantification by Soret absorbance was performed with 50 µg of protein. Pyridine hemochrome assays were performed as previously described (Berry and Trumpower, 1987) in the assay buffer. If needed, sodium dithionite powder was used for protein reduction. Maturation of peptides by CcsBA was assessed by measuring the maximum or minimum of the second derivative of the final reaction spectrum.

In vitro reconstitutions were performed aerobically (HCCS) or anaerobically (HCCS and CcsBA). For anaerobic reactions, all reagents were equilibrated with N₂ (95%) and H₂ (5%) in a Coy anaerobic airlock chamber. Affinity purified synthase (HCCS or CcsBA) was combined with apo equine heart cytochrome c or apo peptide at indicated concentrations. Apo cyt c and peptide concentrations used were determined to be within the range for maximal heme attachment as determined by a titration. An initial spectra and sample for SDS–PAGE analysis were obtained. Five millimolar DTT was added to initiate the reaction. Reactions were placed at 37°C, and spectra and gel samples were taken at indicated time points. Gel samples were immediately placed in loading dye (1:1 v/v) to stop the reaction.

Apocytochrome c preparation was modified from Babul and Stellwagen, 1972. Cytochrome c from equine (horse) heart was obtained from Sigma, and a 1 ml 10 mg/ml solution was prepared in water. To remove heme, 200 µl of glacial acetic acid and 1.5 ml of 0.8% silver sulfate were added and the solution was incubated at 44°C for 4 hr. Sample was dialyzed in 0.2 M acetic acid overnight at 4°C. To precipitate apo cytochrome c and remove silver, sample was transferred to a conical tube and 10 volumes of cold acid acetone were added. Apo cytochrome c was pelleted by spinning at 15,000 rpm for 20 min at 4°C. The pellet was washed with acid acetone and pelleted three times. The apoprotein was resuspended in 0.2M acetic acid (~1 ml), and solid urea was added until the solution turned clear. A 25-fold molar excess of 2-mercaptoethonal was added and incubated at room temperature to remove silver sulfate. Apo cytochrome c was clarified by centrifugation at 12,000 rpm for 10 min at room temperature. Supernatant was dialyzed in 0.2 M acetic acid overnight and buffer exchanged into PBS by concentration in an Amicon Concentrator with 3 kDa molecular weight cutoff. Protein concentration was determined using a BSA standard curve and Coomassie protein staining on SDS–PAGE.

Affinity purified proteins or indicated in vitro reactions were resolved on an Agilent 1100 HPLC system equipped with an Agilent SEC-3 column in the purification or in vitro reaction buffer.

To determine whether in vitro synthesized cytochrome c (or peptide) was released from the synthase, GST-HCCS bound to glutathione agarose (75 µl) was combined with apocyt c (or peptide) under standard in vitro conditions (100 µl volume in addition to the 75 µl of beads). After a 1 hr reaction, the glutathione agarose-bound GST-HCCS were pelleted, and the supernatant was collected. Subsequently, the beads were washed to allow for analysis of protein retained on the beads. The bead fraction and supernatant were separated by SDS–PAGE and heme stained to determine which fraction contained heme attached cyt c. The amount of holo cyt c/peptide matured and released was quantitated using Image J (Rasband, 1997) by determining the ratio of the supernatant derived heme band with the total peptide band signal (beads plus supernatant). The supernatant was further analyzed by UV–vis spectroscopy.

The supernatant from the released product assay (above) was extracted and pooled in an anaerobic environment and then concentrated in a 3K VivaspinTurbo cutoff filter (Sartorius) to obtain 300 μl of 0.4 mg/ml cyt c as determined by heme absorbance at 550 nm (ext. coef. 29.5 mM⁻¹ cm⁻¹). The near UV (500–300 nm) signal was measured on a Jasco J-815 at room temperature in the reaction buffer (20 mM Tris pH 8.0, 100 mM NaCl, 0.02% DDM, 5 mM DTT). The machine sensitivity was 100 mdeg, the data pitch was 0.5 nm, the scanning mode was continuous, the scanning speed was 50 nm/min, the response rate was 1 s, the bandwidth was 1 nm, and five accumulations were taken (Mendez et al., 2017). A blank sample was subtracted. To compare the absorbance of the released assay product to human cyt c, each CD spectra was subtracted from a blank sample and divided by the absorbance of the protein in the CD machine. The samples were overlaid and coincide with each other.

Redox potential of in vitro synthesized equine heart cytochrome c was determined by a modified Massey method as described in Sutherland et al., 2016, with the following modifications: The absorbance change of heme was monitored at the alpha peak at 550 nm (negligible contribution from reference dye) and the reduction of the reference dye, dichlorophenolindophenol, at 636 nm (negligible contribution from heme).

To determine whether the CXXCH containing peptides inhibited maturation of cytochrome c (i.e. heme attachment), a two-step reaction was performed. Step 1: 10 μM GST-HCCS (30% heme occupancy) was combined with 10 μM apo peptide for a 1 hr in vitro reaction. After 1 hr, UV–vis spectra were performed, and a sample was collected for gel analysis. Step 2: 20 μM apo cytochrome c was added to the reaction. After 1 hr, UV–vis spectra were performed and a sample was collected for gel analysis. To determine whether cytochrome c maturation was inhibited or not inhibited by the peptide, heme stain and Coomassie total protein stain were performed.

Assays were performed as in Sutherland et al., 2018b. Detailed methods are provided in Supplemental Methods.

The HCCS structure was produced using Rosetta, which was informed by structural motifs (Robetta) and coevolutionary data (Gremlin) as has been described (Ovchinnikov et al., 2017; Ovchinnikov et al., 2015; Sutherland et al., 2018b; Sutherland et al., 2018a), and will be detailed later.