During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates*

Background: Cytochrome c maturation (Ccm) forms thioether bonds between heme b and c-type apocytochromes. Results: CcmI chaperone exhibits a very high binding affinity for the class I c-type apocytochromes, which decreases drastically in the presence of heme. Conclusion: CcmI holds the c-type apocytochromes tightly until heme coordination yields their b-type intermediates during Ccm. Significance: Interactions between the c-type apocytochromes and chaperones are critical for the Ccm process. The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c2 by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c2 and c1, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm KD values). When present, heme induced conformational changes in class I apocytochromes (e.g. c2) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c2 and that heme converts apocytochrome c2 into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c2) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.

The c-type cytochromes are ubiquitous electron transfer proteins involved in energy transduction in almost all living cells, and they also play critical roles in other cellular pathways (e.g. apoptosis in eukaryotes) (1)(2)(3). These proteins always contain at least one conserved heme-binding site (C 1 XXC 2 H), where heme b (protoporphyrin IX-Fe) is covalently ligated. The stereospecificity of the thioether bonds formed between the vinyl-2 and vinyl-4 of heme and the thiols of Cys 1 and Cys 2 of the heme-binding site, respectively, is universally conserved (4). The His residue of the heme-binding site, together with another Met or His residue, provides axial ligation to heme iron (5). Despite these common features, the c-type cytochromes are diverse in terms of size, three-dimensional structure, heme content, and physicochemical properties. Previously, Ambler (6) grouped the c-type cytochromes into four broad classes. Class I is a large group that includes small, globular, and soluble c-type cytochromes. They usually contain a single N-terminal heme-binding site with a Met residue as the sixth ligand, located near their C termini (e.g. mitochondrial cytochrome c). They are divided into subfamilies according to their structures, functions, and the properties of their cytochrome domains (1,7). Class II c-type cytochromes include the high-spin cytochrome cЈ with a C-terminally located heme-binding motif and a four-helical bundle fold. Class III c-type cytochromes comprise the low E m (redox potential) multi-heme proteins with generally bis-His coordination, and the c-type cytochromes with additional non-heme cofactors (e.g. flavins), such as flavocytochrome c 3, are grouped in class IV.
Rhodobacter capsulatus produces a variety of c-type cytochromes under different growth conditions. These include the class I C-terminally membrane-bound cytochrome c 1 subunit of the cytochrome bc 1 complex (8) and the N-terminally membrane-attached cytochrome c p and c o subunits of the cbb 3 -type oxygen reductase (9,10), as well as the soluble cytochrome c 2 and the N-terminally membrane-attached cytochrome c y as electron carriers (11,12). The class II soluble high-spin cytochrome cЈ is involved in NO detoxification (13), and the class III membrane-attached pentaheme c-type cytochrome DorC con-veys electrons from the Q/QH 2 pool to dimethyl sulfoxide (DMSO) 3 reducing it to dimethylsulfide (14).
R. capsulatus and other ␣and ␥-proteobacteria, archaea, and mitochondria of plants and red algae carry out the process of covalent heme ligation to the c-type apocytochromes via a membrane complex, designated as cytochrome c maturation (Ccm) System I (15)(16)(17)(18). The overall process relies on several cellular pathways, including post-translational modification and secretion of c-type apocytochromes and the folding and degradation of proteins, as well as maintenance of a suitable thioredox environment conducive to cofactor insertion. The Ccm complex involves nine membrane proteins (CcmABCDEFGHI) that are responsible for the chaperoning of c-type apocytochromes and heme as well as their covalent ligation (15).
CcmI is composed of two different domains and forms with CcmF and CcmH a multisubunit protein complex responsible for heme ligation (19 -23). The N-terminal CcmI-1 domain is membrane-integral via two transmembrane helices and has a cytoplasmic loop with a leucine zipper-like motif. The large periplasmic C-terminal CcmI-2 domain contains three tetratricopeptide repeats (TPR) (20, 24 -26). TPR domains are involved in protein-protein interactions and form two anti-parallel ␣-helices packed in tandem arrays as a superhelical structure with a convex and a concave surface where the target proteins bind (27). Genetic studies indicate that the CcmI-1 domain of R. capsulatus is required for the maturation of all c-type cytochromes, whereas some amount of C-terminally membrane-anchored cytochrome c 1 is produced in the absence of CcmI-2 (20). Recently we (25) and others (28,29) showed that CcmI binds as a chaperone the C-terminal helix of apocytochrome c 2 , primarily via its TPR-containing CcmI-2 domain.
CcmE is a heme-handling membrane protein with a ␤-barrel domain and a flexible C-terminal stretch (30 -32). It binds covalently vinyl-2 of heme b through a surface-exposed His residue at its conserved HXXXY site (33)(34)(35). HoloCcmE formation and delivery of heme to c-type apocytochromes rely on a specific ABC-type transporter complex (CcmABCD). Once apoCcmE is heme-loaded, an ATP hydrolysis-dependent conformational change (36) renders it competent to deliver heme to c-type apocytochromes. Recently, we found that apoCcmE interacts with the N-terminal heme-binding region of apocytochrome c 2 and forms a ternary complex together with CcmI in vitro (37). Moreover, in R. capsulatus membrane fractions, apoCcmE also interacts with both CcmI and CcmH (37). In addition, holoCcmE is known to form a complex with CcmF in Escherichia coli (38). Altogether, these findings indicate that the heme ligation complex, CcmFHI, contains CcmE and CcmG, possibly forming a large "maturase supercomplex" (15).
In this study, we investigated the binding interactions among CcmI, apoCcmE, and different c-type apocytochromes that are distinct from apocytochrome c 2 in order to understand how R. capsulatus Ccm System I matures many structurally dissim-ilar c-type cytochromes. We also explored for the first time how the availability of heme affects these chaperone-apocytochrome interactions. We found that CcmI and apoCcmE bind different c-type apocytochromes with markedly different affinities (K D values) and that the strength of these interactions does not correlate with the distinct secondary structures. Remarkably, heme modulates these binding interactions significantly, suggesting that CcmI holds the c-type apocytochromes tightly until their intermediate b-type derivatives are formed. We propose that these intermediates are subsequently converted into mature c-type cytochromes upon completion of covalent heme ligation by the remaining components of the Ccm complex.

Experimental Procedures
Bacterial Strains and Growth Conditions-The bacterial strains and plasmids used in this work are described in Table 1. E. coli strains were grown aerobically at 37°C in Luria-Bertani broth medium supplemented with ampicillin (100 g/ml). Cultures were induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside (25). R. capsulatus strains were grown chemoheterotrophically (i.e. by respiration) at 35°C on MPYE (mineralpeptone-yeast-extract) enriched medium supplemented with tetracycline or spectinomycin at 2.5 or 10 g/ml, respectively (39).
Molecular Genetic Techniques-Apocytochromes c 1 and cЈ and their derivatives were produced as done earlier for apocytochrome c 2 (25). R. capsulatus native cytochrome c 1 (petC) has four Cys residues (Cys-34 and Cys-37 of the C 1 XXC 2 H hemebinding site and Cys-144 and Cys-167, which form a disulfide bond) (39). Mutating the latter two Cys residues of cytochrome c 1 does not affect its maturation but lowers its E m and renders it nonfunctional. An additional mutation, A181T, in the heme environment corrects this defect to yield a fully functional cytochrome c 1 variant (40). This disulfide-less apocytochrome c 1 , chosen to avoid complications that could arise from the extra Cys residues, was considered the "wild type" for maturation purposes. Two different apocytochrome c 1 derivatives (pMAM1 and pMAM2) were constructed by PCR amplification using the mutant petC allele on plasmid pPET1-C144A/ C167A/A181T (40) as a template and the primers NdeI-Cytc 1 -Fw (inserting a NdeI restriction site at the 5Ј-end and removing the cytochrome c 1 signal sequence) and Cytc 1 _ BamHI-Rv or Cytc 1 t39_BamHI-Rv (inserting a BamHI restriction site either at the 3Ј-end of petC or 117 bp upstream of its stop codon, respectively) ( Table 2). The PCR products were cloned into the same restriction sites in pCS1302 (23) to yield N-terminally Strep II-tagged signal sequence-less apocytochrome c 1 derivatives with a Factor Xa cleavage site for tag removal. Plasmid pMAM1 encoded a soluble variant of apocytochrome c 1 lacking its C-terminal 39-amino acid-long membrane anchor (apocytochrome c 1 t39), and pMAM2 encoded full-length apocytochrome c 1 (Table 1). Similarly, a signal sequence-less and N-terminally Strep II-tagged apocytochrome cЈ (RCC02682 corresponding to cycP) was obtained by PCR amplification using R. capsulatus chromosomal DNA as a template and the primers NdeI-cЈ-Fw and cЈ-BamHI-Rv containing the NdeI and BamHI restriction sites, yielding plasmid pAV6 after its cloning into pCS1302 (Tables 1 and 2). In addi-tion, a mature form of Hydrogenobacter thermophilus cytochrome c 552 was obtained by PCR amplification using plasmid pCS1208 4 (Table 1) as a template and primers Htssdel-Fw (inserting an NdeI site immediately downstream of its signal sequence) and CS46 (located 3Ј of the BamHI restriction site) ( Table 2). The PCR product was cloned into pCS1302 to yield pAV5, producing Ht-apocytochrome c 552 lacking its signal sequence. Plasmid pAV5C13SC16S containing the double Cys to Ser substitutions at the heme-binding site of Ht-apocytochrome c 552 was derived from pAV5 using a QuikChange sitedirected mutagenesis kit (Invitrogen) and Htc 552 C13/C16-Fw and Htc 552 C13/C16-Rv primers (Tables 1 and 2) according to the supplier's recommendation. All constructs were analyzed by serial cloner 2.1 and confirmed by DNA sequencing.
Protein Purification-The proteins His 10 -CcmI, His 10 -CcmI-2, His 10 -apoCcmE, and FLAG-CcmI were purified by affinity chromatography using Ni-Sepharose high performance (GE Healthcare) and anti-FLAG M2 affinity (Sigma) resins, respectively (25,37). Strep-tagged c-type apocytochromes were purified as described earlier (25). The Cys-less variant of H. thermophilus cytochrome c 552 , Ht-apocytochrome b-c 552 , was incubated overnight with 50 mM Tris-HCl, 50 mM NaCl, and 1 M imidazole for heme removal. The imidazole displaced the heme axial ligands, leading to precipitation of the heme, which was removed by centrifugation at 14,000 ϫ g for 15 min, originating Ht-apocytochrome b-c 552 . Purified protein samples were checked by SDS-PAGE for purity (Ͼ95%), concentrated by ultrafiltration, and desalted using PD-10 columns (GE Healthcare). A synthetic peptide carrying a Strep II tag and Factor Xa cleavage site, corresponding to cytochrome c 1 residues 222-241, was produced by Thermo Fisher Scientific.
Protein-Protein Interactions Monitored by Co-purification Assays-Direct interactions among His 10 -apoCcmE, FLAG-CcmI, and different Strep-tagged c-type apocytochromes were assayed as described previously (25). Briefly, equimolar amounts of Strep-tagged c-type apocytochromes (ϳ1 M) were mixed with substoichiometric amounts of His 10 -apoCcmE or FLAG-CcmI (ϳ0.1 M) in the assay buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0, final volume of 400 l) and incubated for 2 h at 25°C with gentle shaking. The mixture was loaded onto a mini (200 l volume) Strep-Tactin resin column equilibrated with the same buffer. The column was washed extensively with 2 ml of assay buffer (10 column volumes) and eluted with the same buffer containing 2.5 mM desthiobiotin. Flow-through and elution fractions were precipitated with methanol:acetone (7:2, v/v) overnight at Ϫ20°C, and interacting partners were analyzed by SDS-PAGE. Binding assays using the synthetic peptides instead of the c-type apocytochromes followed a similar protocol. As appropriate, different amounts of hemin (i.e. heme chloride) (Frontier Scientific Inc.) dissolved in DMSO (determined using the extinction coefficient of 179 cm Ϫ1 mM Ϫ1 at 400 nm in 40% DMSO (41)) were added to the incubation mixtures.
Protein-Protein Interactions Monitored by Biolayer Interferometry-The binding kinetics of His 10 -CcmI and His 10 -apoCcmE to different Strep-tagged c-type apocytochromes was monitored quantitatively in real time by biolayer interferometry (BLI) using an Octet RED96 instrument (ForteBio). Purified c-type apocytochromes (i.e. ligands) were biotinylated using the EZ-Link TM NHS-PEG 4 biotinylation kit (Thermo Scientific) to immobilize them on streptavidin-coated biosensors (SA-sensors). SA-sensors were loaded with biotinylated c-type apocytochromes (Bt-apocytochromes) by soaking them in a buffer containing the desired Bt-apocytochrome at ϳ400 nM, 50 mM Tris-HCl, pH 8, 100 mM NaCl, 0.01% n-dodecyl-␤-D-maltoside, and 1% BSA at 30°C and 1000 rpm with shaking. A reference sensor was dipped into a well containing the assay buffer and lacking the Bt-apocytochrome to assess nonspecific binding of the analyte (CcmI or apoCcmE) to the sensor. After the SA-sensors were washed with the same buffer, unoccupied residual 4 C. Sanders and F. Daldal, unpublished observations.

TABLE 1 Strains and plasmids used in this work
Res and Ps refer to respiratory and photosynthetic growth, respectively; Nadi refers to cytochrome c oxidase-dependent catalysis of ␣-naphtol to indophenol blue. R. capsulatus MT1131 is referred to as the wild type with respect to its c-type cytochrome profile and growth properties.

Specificity of CcmI for Different Classes of Apocytochromes
streptavidin sites on the SA-sensors were blocked with biocytin (10 g/ml), and following another washing step, a baseline signal was recorded. Different Bt-apocytochrome-loaded SA-sensors were incubated with increasing concentrations of analyte (i.e. CcmI from 4 nM to 30 M or apoCcmE from 0.3 to 20 M) (association step). Subsequent washing of the biosensors with the assay buffer released the analyte (CcmI or apoCcmE) from the immobilized ligand (dissociation step). An assay lacking the analyte was used as a negative control to confirm that the observed shifts were due to the ligand-analyte complexes.
The collected data were used to determine the kinetic parameters. The range of concentrations used depended on the Btapocytochrome tested to obtain data under nonsaturating binding conditions. Higher concentrations of CcmI or apoCcmE were needed in the case of class II apocytochrome cЈ, which enhanced nonspecific binding to the sensors. To mitigate this problem, the n-dodecyl-␤-D-maltoside concentration of the wash buffer was increased to 0.05%. The kinetics performed in the presence of heme used FLAG-CcmI instead of His 10 -CcmI as an analyte to avoid possible binding of heme to the His 10 epitope. In addition, the standard assay buffer containing BSA (known to bind heme; see Ref. 42) was substituted with 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.01% Tween-20. Under these conditions, hemin was used at concentrations ranging from 0.1 to 6.4 M to monitor its binding to apocytochrome c 2 . To investigate the effect of heme on CcmI-apocytochrome c 2 interactions, we first repeated full kinetic measurements using this buffer and FLAG-CcmI at concentrations ranging from 0.7 to 180 nM. Then, the assay buffer was supplemented with 2 M hemin to yield a new baseline (accounting for binding of heme to apocytochrome c 2 ). Increased FLAG-CcmI concentrations (from 0.07 to 2.4 M) were used to account for decreased apparent association responses. The k on and k off rates of binding and the K D values for each interacting pair were determined by fitting the experimental data to 1:1 homogenous or 2:1 heterogeneous kinetic models describing bimolecular interactions according to the manufacturer's literature (Forte-Bio) (43). The quality of the fit between the experimental and calculated data was evaluated according to the following parameters: error values for k on and k off (at least an order of magnitude lower than the k values), residual values (Ͻ10% of the maximum response of the fitting curve), R 2 (Ͼ0.95) and X 2 (Ͻ3) (43). SDS-PAGE and Immunoblot Analyses-SDS-PAGE under reducing conditions (5% ␤-mercaptoethanol) was performed using 15% gels according to Laemmli (44), and covalently bound heme-containing proteins were detected using tetramethylbenzidine (TMBZ) as described elsewhere (45). For apocytochrome cЈ immunodetection, gel-resolved proteins were electroblotted onto Immobilon-P PVDF membranes (Millipore) and probed with rabbit polyclonal antibodies specific for R. capsulatus cytochrome cЈ (a kind gift of Dr. R. Prince). Horseradish peroxidase-conjugated anti-rabbit IgG antibodies (GE Healthcare) were used as secondary antibodies, and detection was performed using SuperSignal TM West Pico chemiluminescent substrate from Thermo Scientific.
Circular Dichroism Spectroscopy-The far-UV circular dichroism (CD) spectra (195-240 nm) were recorded with a model 202 spectropolarimeter (AVIV Biomedical, Inc) using a 2-mm-path length cuvette (Hellma, Inc.) as done previously (25). The CD spectra of proteins (15 M) in 20 mM sodium phosphate buffer, pH 7.5, were recorded using a 3-nm bandwidth, a 2-nm step size, and a time constant of 10 s. The CD spectrum of the buffer was subtracted from the spectra of the proteins, and the absorbance values were converted to the mean residue ellipticity [] (deg cm 2 dmol Ϫ1 ) at each wavelength using the relation [] ϭ /(10 ϫ C ϫ n ϫ l), where is the observed ellipticity in millidegrees at wavelength , C is the molar protein concentration, n is the number of amino acids of the protein, and l is the path-length of the cuvette in cm. The CD spectra monitoring the effect of hemin on apocytochrome c 2 or CcmI or their interactions were recorded using 1-nm step size and 20 mM sodium phosphate buffer, pH 7.5, supplemented with 10 mM potassium cyanide to prevent hemin aggregation. Hemin stock solution was prepared in 100 mM NaOH, and the concentration was determined using the extinction coefficient of 5.84 ϫ 10 4 cm Ϫ1 M Ϫ1 at 385 nm in the same solution (46). The spectra were recorded 2 h after hemin addition to ensure its complete binding to apocytochrome c 2 and that full conformational changes had been induced under oxidizing conditions. The CD spectra of the buffers (with or without hemin) were subtracted from the spectra of the proteins, and the absorbance values were converted into the mean residue ellipticity [] (deg cm 2 dmol Ϫ1 ) as described above. To determine the effect of heme on apocytochrome c 2 -CcmI interactions, the protein mixtures (molar ratio of apocytochrome c 2 to CcmI, 2:1) were incubated for 2 h at room temperature without or with hemin (at 2-8-fold molar excess of apocytochrome c 2 ), and their CD spectra were compared with the sum of the spectra of individual proteins obtained under the same conditions after subtraction of the spectral contributions of the corresponding buffers.

Reconstitution of b-Type Cytochrome
Intermediates-A stoichiometric amount of hemin dissolved in DMSO was added slowly from a stock solution of 1 mM to 10 M c-type apocytochrome in 50 mM Tris-HCl, 150 mM NaCl, pH 8. The sample was stirred for 5-10 min between each addition to reach equilibrium, and visible spectra between 380 and 650 nm were taken to monitor the binding of hemin to the c-type apocytochromes. Unbound hemin was removed by size exclusion chromatography (PD-10 column, GE Healthcare), and after concentration, the visible spectra of the newly formed b-type cytochromes were recorded as prepared (air-oxidized) and after dithionite reduction. The relative amounts of reconstituted b-type cytochrome derivatives of R. capsulatus c-type apocytochromes were determined by taking as 100% the amount of Ht-apocytochrome b-c 552 reconstituted under the same conditions.
Chemicals-All chemicals and solvents were of high purity and HPLC spectral grades and were purchased from commercial sources.

Results
Overproduction and Purification of CcmI, apoCcmE, and c-Type Apocytochromes-We showed previously that R. capsulatus CcmI binds tightly to the C-terminal helix, whereas apoCcmE interacts with the N-terminal heme-binding region of apocytochrome c 2 (25,37). As this bacterium produces various c-type cytochromes, in the current work we inquired whether these interactions were exclusive to apocytochrome c 2 or more general, including other c-type apocytochromes. Considering that maturation of cytochrome cЈ in R. capsulatus had not been examined earlier, we first analyzed soluble extracts of R. capsulatus mutants lacking CcmI or CcmE (MT-SRP1 (20) or MD2 (47), respectively) using SDS-PAGE/ TMBZ staining and immunodetection with cytochrome cЈ antibodies. These mutants lacked cytochrome cЈ (Fig. 1), confirming that CcmI and CcmE of Ccm System I were required for its maturation. Based on these findings, we chose in addition to apocytochrome c 2 the class I membraneanchored cytochrome c 1 , for which maturation is independent of the CcmI-2 domain of CcmI, and the class II-soluble cytochrome cЈ, which has a nonglobular three-dimensional structure ( Fig. 2A).
CcmI Discriminates among Different Classes of c-Type Apocytochromes-The chaperone activity of CcmI against the different classes of c-type apocytochromes was probed first using co-purification assays under the conditions established previously for apocytochrome c 2 (25). SDS-PAGE analyses of elution fractions showed that different amounts of CcmI co-purified with different c-type apocytochromes (Fig. 3A). Semiquantitative image analyses of Coomassie-stained gels estimated that CcmI co-purified with apocytochrome c 1 at about 70% (Fig. 3A, lane 4) of the amount of CcmI retained by apocytochrome c 2 (lane 2). This decrease in CcmI retention was more obvious with the soluble apocytochrome c 1 t39 variant (Fig. 3A, lane 5). Remarkably, no detectable amount of CcmI co-purified with apocytochrome cЈ (Fig. 3A, lane 3). We concluded that CcmI associates more readily with the class I than the class II c-type apocytochromes under the conditions used.
Using a full-length apocytochrome c 1 and its membrane-anchorless variant, apocytochrome c 1 t39, we next probed the role of the different domains of CcmI in vitro. Co-purification assays conducted using intact CcmI or only its CcmI-2 domain with full-length or truncated apocytochrome c 1 derivatives showed that the full-length apocytochrome c 1 interacted better with CcmI than the truncated apocytochrome c 1 t39. Also, the amount of CcmI-2 that co-purified with either derivative of apocytochrome c 1 was higher than CcmI (Fig. 3B). These findings suggested that neither the membrane anchor of apocytochrome c 1 nor the CcmI-1 domain (i.e. the first transmembrane helix and the adjacent leucine zipper-containing cytoplasmic loop absent in the CcmI-2 derivative used) of CcmI is essential for these interactions in vitro. Lastly, using a synthetic peptide (NH 2 -WSHPQFEKIEGRTVDQMAQVDSAFLMWAAEPK-COOH) corresponding to the C-terminal helix of apocytochrome c 1 t39 (i.e. the C-terminal helix that interacts with the N-terminal heme-binding helix), we tested whether CcmI would recognize this helical sequence of apocytochrome c 1 , as observed elsewhere with that of apocytochrome c 2 (25). Incubation of increasing amounts (10 and 20 g) of this peptide with purified CcmI led to a concentration-dependent co-purification of the CcmI-peptide complex (Fig. 3C). The amount of CcmI co-purified was lower than that observed with the same amount of the apocytochrome c 2 peptide used previously (25), paralleling the findings of the binding assays using apocytochromes c 1 and c 2 (Fig. 3A). The data indicated that in all class I c-type apocytochromes the C-terminal helix, which is orthogonal to the N-terminal heme binding site-containing helix, is sufficient to promote binding to CcmI. ApoCcmE Recognizes Differently Class I and Class II c-Type Apocytochromes-In this work, we extended the apoCcmEapocytochrome c 2 binding studies carried out earlier (37) to other c-type apocytochromes and found that apoCcmE, like CcmI, binds apocytochrome c 1 but not apocytochrome cЈ (Fig.  4A). Semiquantitative image analyses of Coomassie-stained gels revealed that the amounts of apoCcmE co-purifying with apocytochrome c 1 decreased to 60% of that seen with apocytochrome c 2 (Fig. 4A, lanes 1 and 3), whereas no detectable interaction was seen with apocytochrome cЈ (lane 2). Moreover, a comparison of the truncated apocytochrome c 1 t39 with its Cysless derivative (Cys to Ser substitutions at the heme-binding site) indicated that the occurrence of a disulfide bond at the heme-binding site had no effect on the apoCcmE-apocytochrome c 1 t39 interactions (Fig. 4B), unlike apocytochrome c 2 (37).
Binding Kinetics of Various c-Type Apocytochromes to CcmI and apoCcmE-Using BLI, the binding affinities of CcmI and apoCcmE to c-type apocytochromes were quantified by realtime binding assays. The association (k on )/dissociation (k off ) rates and the binding affinity (K D ) constants of the appropriate protein couples were determined as described under "Experimental Procedures." Negative controls lacking the analytes confirmed that the observed interference shifts originated from the ligand-analyte complexes. Using Octet data analysis software (ForteBio), experimental data (association and dissociation curves) were fit to a homogeneous 1:1 bimolecular proteinprotein interaction model, and the binding parameters (k on , k off , and K D ) of the ligand-analyte couple were determined (Table 3).
With the CcmI-Bt-apocytochrome c 2 complexes, the association curves exhibited rapid increases until reaching equilibrium, and the dissociation curves followed slow decay kinetics (monitored for longer time periods for better data collection) (Fig. 3D). This behavior reflected fast binding of CcmI to apocytochrome c 2 to form a stable complex. Similar experiments conducted with other c-type apocytochromes indicated that CcmI interacted strongly with the class I apocytochromes c 1 and c 1 t39 tested ( Table 3, K D values (nM range)). In contrast, although CcmI associated rapidly with apocytochrome cЈ, it also dissociated rapidly, indicating that it bound weakly to this class II c-type apocytochrome ( Table 3, K D values (M range)). Thus, the binding kinetics confirmed and quantified the findings of the co-purification assays (Fig. 3A). In all cases, the k on rates were comparable, indicating that the c-type apocytochromes bind rapidly to CcmI, but the k off rates were faster for unstable (i.e. apocytochrome cЈ) and slower for the stable (i.e. apocytochromes c 2 , c 1 , and c 1 t39) binary complexes.
Next, the binding of apoCcmE to c-type apocytochromes was examined using a similar approach (Fig. 4C). The kinetic data showed that apoCcmE associated with apocytochrome c 2 or c 1 t39 at rates slower than those seen with CcmI and dissociated at faster rates, yielding K D values in the micromolar range (Table 3). Furthermore, even though apoCcmE bound apocy-  The amount of CcmI that co-purified with apocytochrome c 2 was taken as 100% for semiquantitative estimation using ImageJ software and was compared with those seen with other c-type apocytochromes. Although the samples were reduced with ␤-mercaptoethanol, homodimers of apocytochrome c 1 t39 (marked as ⅐, above CcmI) were observed when its heme-binding site Cys residues were intact. B, co-purification of FLAG-CcmI and its derivative, His 10 -CcmI-2, with apocytochrome c 1 and its truncated derivative, apocytochrome c 1 t39. The amount of CcmI that co-purified with apocytochrome c 1 (lane 3) was taken as 100%, and that of the other c-type apocytochromes was determined as described above.  (Table 3). Cys/Ser*

FIGURE 4. ApoCcmE differently recognizes class I and class II c-type apocytochromes.
A, co-purification of His 10 -apoCcmE with stoichiometric amounts of different c-type apocytochromes. The amounts of apoCcmE co-purified with apocytochrome c 2 (lane 1) was taken as 100% and compared with those seen with apocytochromes cЈ and c 1 (lanes 2 and 3, respectively). B, co-purification of His 10 -apoCcmE with apocytochrome c 1 t39 and with its Cys-less derivative, apocytochrome c 1 t39Cys/Ser*. The amounts of ApoCcmE co-purified with apocytochrome c 1 t39 (lane 1) and its Cys-less derivative (Cys/Ser*) were determined as described in the legend for Fig. 3. C, real-time protein-protein interactions between Strep-Bt-apocytochrome c 2 immobilized on a SA-biosensor (ligand) and His 10 -apoCcmE (analyte). The aligned sensorgram traces showing baseline (B) followed by association (A) and dissociation (D) steps were obtained using 400 nM Bt-apocytochrome c 2 and varying concentrations of apoCcmE. The raw data were fitted with high accuracy to a homogeneous 1:1 bimolecular interaction model, and the kinetic parameters were determined (Table 3).
tochrome cЈ at rates similar to apocytochromes c 2 or c 1 t39, it dissociated rapidly, showing ϳ100-fold higher K D values. Overall the data established that both CcmI and apoCcmE recognized the class I c-type apocytochromes with higher affinities than their class II counterparts.
Probing the Secondary Structures of Various c-Type Apocytochromes Using CD Spectroscopy-The secondary structures (globular class I cytochromes c 2 and c 1 versus four-helical bundle class II cytochrome cЈ) of different c-type apocytochromes were examined by CD spectroscopy in the far-UV region (Fig.   5A). The CD spectra of apocytochromes c 2 and cЈ exhibited a negative peak around 203 nm and low ellipticity below 215 nm, showing their random coil conformations, respectively. In contrast, both apocytochrome c 1 and apocytochrome c 1 t39 exhibited CD spectra more characteristic of ␣-helical proteins, with two negative peaks at 208 and 223 nm. Interestingly, however, the two class I apocytochromes, c 2 and c 1 , which have similar globular folds in their mature forms, exhibited different secondary structures in their apocytochrome forms (48,49). In addition, the R. capsulatus class II apocytochrome cЈ also differed from its E. coli homologue, Cyt b 562 , which forms a molten globule in the absence of heme (50), even though both holocytochromes have four-helical bundle structures. Finally, the CD spectra of CcmI and CcmI-2 showed the characteristics of ␣-helical proteins (Fig. 5B).
Release of Apocytochrome c 2 from CcmI Is Facilitated by the Presence of Hemin-The observed tight binding of CcmI to class I c-type apocytochromes was remarkable, leading us to probe whether heme affected these interactions. This was addressed by choosing cytochrome c 2 as a prototype for class I c-type apocytochrome. We reasoned that if heme induces the formation of a b-type derivative of apocytochrome c 2 , with a molten globule-like structure (reminiscent of that of mature cytochrome c 2 ), then this intermediate might not bind CcmI tightly, similar to what we observed earlier with a native form (25). To test this hypothesis, we first investigated the kinetics of heme binding to Bt-apocytochrome c 2 using BLI. The data showed that heme bound to, and dissociated from, apocytochrome c 2 rapidly (Fig. 6A). A 1:1 bimolecular kinetic model indicated that the apocytochrome c 2 -heme complex had an affinity constant (K D ) in the order of 1 M and was not very stable (Table 3). A similar low affinity has been reported for the horse heart apocytochrome c-heme complex under oxidizing conditions (46). Next, we examined the effects of heme on the CD spectra of apocytochrome c 2 and CcmI. Upon the addition  of hemin, the far-UV CD spectrum of apocytochrome c 2 changed drastically, with increased helical content, paralleling increased amount of hemin (Fig. 6B). On the other hand, the CD spectrum of CcmI was hardly affected, with only minor spectral changes seen in the presence of excess (16-fold) hemin (Fig. 6C).
For the effect of heme on CcmI-apocytochrome c 2 interactions, we first tested the co-purification of CcmI with apocytochrome c 2 in the presence of hemin (Fig. 7A). The addition of stoichiometric amounts of hemin (ϳ1 M) decreased the amount of CcmI that co-purified with apocytochrome c 2 by ϳ25% (Fig. 7A). Next, the binding kinetics of CcmI to apocytochrome c 2 were monitored by BLI in the absence of hemin but omitting BSA and replacing His 10 -CcmI with FLAG-CcmI to minimize spurious heme interference. As above, we observed K D values in the order of nanomolar for CcmI-apocytochrome c 2 interactions for these derivatives (Table 3). However, when the assays were repeated in the presence of 2 M hemin (ϳ5 fold molar excess of Bt-apocytochrome c 2 ), the association and dissociation kinetics could not fit reliably to a 1:1 homogeneous model, suggesting the presence of a nonhomogeneous ligand population. In cases where "active" and "inactive" forms of ligands toward the analyte are expected, the use of a 2:1 heterogeneous model becomes appropriate. Indeed, upon the addition of heme, a fraction of apocytochrome c 2 yielded a noncovalent heme-containing b-type cytochrome derivative (see below). When the kinetic data were fitted to a 2:1 heterogeneous model, two different K D values (with acceptable X 2 and R 2 values) for CcmI-apocytochrome c 2 interactions were deduced (Table 3). These values were 15-20 times higher than the K D seen in the absence of hemin, clearly indicating that the CcmI-apocytochrome c 2 interactions had weakened. Previously, CD spectroscopy had shown that CcmI and apocytochrome c 2 change their conformations upon binding to each other in the absence of hemin (25). This approach was used to further document the effect of heme on CcmI-apocytochrome c 2 interactions. The secondary structure changes were monitored after incubating CcmI and apocytochrome c 2 in the presence (molar excess) or absence of hemin, and the CD spectra obtained were compared with the sums of the spectra of the individual proteins recorded under the same experimental conditions (Fig. 7B). These comparisons showed that the conformational changes induced by the apocytochrome c 2 -CcmI interactions decreased markedly in the presence of hemin. This finding further supported the view that a fraction of apocytochrome c 2 changed its secondary structure upon binding to heme and weakened its interactions with CcmI, lowering the CD-detected spectral changes (Fig. 7C).
The c-Type Apocytochromes Can Form b-Type Cytochrome Variants in the Presence of Hemin-Optical spectroscopy was used to monitor noncovalent binding of heme to apocytochrome c 2 and to other selected c-type apocytochromes to assess whether indeed they form b-type cytochrome variants. As a control for formation of a b-type cytochrome variant from a c-type apocytochrome, a Cys-less derivative of H. thermophilus cytochrome c 552 was used. When expressed in E. coli cyto-plasm, native H. thermophilus cytochrome c 552 contains covalently ligated heme (51) even in the absence of the Ccm System I and under aerobic growth conditions. Similarly, the Cys-less derivative of H. thermophilus cytochrome c 552 produces a noncovalent heme-containing b-type cytochrome (called cytochrome b-c 552 ) (52). Overnight incubation of purified cytochrome b-c 552 in the presence of 1 M imidazole displaces its heme to yield apocytochrome b-c 552 (52) (data not shown). We purified these variants of H. thermophilus cytochrome c 552 , and SDS-PAGE/TMBZ analyses confirmed the absence of covalently bound heme in both cytochrome b-c 552 and its corresponding apocytochrome b-c 552 (data not shown). The CD spectra of these proteins resembled those of typical ␣-helical proteins with the amounts of secondary structures being increased from apocytochrome b-c 552 to cytochrome b-c 552 to cytochrome c 552 (data not shown). The binding of heme enhanced the secondary structure formation, even though apocytochrome b-c 552 already had some secondary structure in the absence of heme as compared with the apocytochromes c 2 and cЈ (Fig. 5A). Thus, H. thermophilus cytochrome c 552 and its derivatives provided valid controls for the formation of b-type cytochrome from the c-type apocytochromes. The addition of stoichiometric amounts of hemin to R. capsulatus c-type apocytochromes resulted in changes in their visible spectra over time. After 30 min of incubation with hemin, apocytochromes c 2 and c 1 , but not apocytochrome cЈ, exhibited spectral features that are typical of b-type cytochromes, with Soret and ␣-bands at 422 and 557 nm in apocytochrome c 2 and at 425 and 559 nm in apocytochrome c 1 , respectively (Fig. 8). Under the conditions where full (100%) incorporation (as confirmed by comparison with similar amount of cytochrome b-c 552 purified from E. coli) of heme to apocytochrome c 552 occurred to yield cytochrome b-c 552 , ϳ20% of the available heme was reconstituted into apocytochrome c 2 , ϳ12% into apocytochrome c 1 , and no detectable amount into apocytochrome cЈ (assuming similar extinction coefficients for all b-type cytochrome derivatives). These findings showed that a fraction of apocytochrome c 2 was converted to its b-type cytochrome derivative in the presence of hemin. Moreover, apocytochromes c 2 and c 1 behaved in opposing ways with respect to the amounts of ellipticity they exhibited and the b-type cytochrome variants they yielded. Thus no direct correlation was seen between the helical content of a c-apocytochrome and its ability to bind heme to yield a b-type cytochrome variant in vitro.

Discussion
Previously, we showed that the chaperone protein CcmI binds to the C-terminal helix of apocytochrome c 2 , whereas the heme-handling protein CcmE recognizes its heme-binding site (25,37). In this work we addressed the next question, which is whether CcmI also chaperones other soluble and membranebound c-type apocytochromes in addition to apocytochrome c 2 . To this end, we chose the class I membrane-anchored apocytochrome c 1 because the TPR motif-containing portion (CcmI-2) of CcmI is not essential for its maturation (20). We also investigated the soluble class II cytochrome cЈ, which has a different (four-helical bundle versus globular) structure than The amounts of the interacting partners were as described under "Materials and Methods," and semiquantitative comparisons (as described in Fig. 3) showed that CcmI-apocytochrome c 2 interactions were weakened, but not completely abolished, in the presence of hemin. B, effect of heme on the CD spectra of the CcmI-apocytochrome c 2 mixture in the absence or presence of hemin (20 M). Spectra were recorded after 2 h of incubation to ensure that all spectral changes were complete. exp and sum, refer to the experimental and calculated spectra, respectively. The calculated spectra were obtained by summing the spectra of each protein alone in the absence or presence of hemin (see Fig. 5, B and C). C, difference spectra between the experimental spectra of CcmI-apocytochrome c 2 minus the calculated spectra shown in B in the absence or presence of hemin (20 M). Decreased conformational changes were seen upon binding of apocytochrome c 2 to CcmI in the presence of hemin, suggesting decreased binding interactions.
the class I members. The production in E. coli of cytochrome cЈ from some species requires co-expression of the Ccm genes (53,54), whereas that from others (e.g. Hydrogenophilus thermoluteolus cytochrome cЈ) does not (54). At the onset of this work, whether cytochrome cЈ maturation in R. capsulatus relied on Ccm System I was unknown (20). Our data showed that cytochrome cЈ maturation in R. capsulatus requires at least CcmE and CcmI and established it as a substrate for Ccm System I. Co-purification and real-time binding (BLI) assays demonstrated that both CcmI and apoCcmE distinguish different classes of c-type apocytochromes and that heme strongly affects these interactions.
Binding of CcmI and apoCcmE to Class I and Class II c-Type Apocytochromes in the Absence of Hemin-Real-time binding studies indicated for the first time that R. capsulatus class I c-type apocytochromes (c 2 , c 1 , and its anchor-less derivative, c 1 t39) had very high (ϳnM range K D ), whereas the class II apocytochrome cЈ had much lower (ϳM range K D ) binding affinities for CcmI (Table 3 and Fig. 9). How the c-type cytochromes interact with their chaperones has not yet been well studied. Only a single experiment, using the soluble portion of Pseudomonas aeruginosa CcmI and the class I apocytochrome c 551 (or a dansylated peptide corresponding to its C-terminal end), has reported a low equilibrium binding K D (ϳϾ100 M) (29). However, a different experimental approach was used in those studies, which renders a direct data comparison difficult. Thus, the reason that different K D values were obtained here and in that study remains unclear.
A major structural determinant of apocytochrome c 2 for binding CcmI is its most C-terminal helix, the equivalent of which is conserved in most class I c-type cytochromes (55). This helix packs orthogonally against the heme-binding site containing N-terminal helix in mature c-type cytochromes. The presence of a hydrophobic molecule (e.g. a porphyrin ring) induces conversion of a random coiled c-type apocytochrome to a molten globular structure as a folding intermediate (56). This promotes interactions between its N-and C-terminal helices. In cytochrome c 1 , unlike the other R. capsulatus c-type cytochromes (i.e. c 2 , c y , c o , and c p ), this "C-terminal helix" precedes its most C-terminal membrane-anchoring helix but becomes the most C-terminal helix in the truncated apocytochrome c 1 t39 derivative. The availability of these two variants allowed us to probe the role of the anchoring and the C-terminal helix of cytochrome c 1 on binding CcmI. Earlier genetic studies indicated that the CcmI-2 portion of CcmI is unnecessary for cytochrome c 1 production, inferring that enough interactions occur during maturation between the Ccm System I and membraneanchored apocytochrome c 1 (20). The in vitro data obtained here further complemented these findings and showed that roughly similar K D values were observed for binding the native or truncated forms of apocytochrome c 1 to CcmI (Table 3). Moreover, co-purification assays showed that CcmI-2 binds both derivatives of apocytochromes c 1 . Thus, the structural determinants recognized by CcmI-2 must also be present in the anchor-less apocytochrome c 1 t39. Indeed, similar to apocytochrome c 2 , a peptide corresponding to the C-terminal helix preceding the membrane anchor of cytochrome c 1 is readily recognized by CcmI (Fig. 3C). Therefore, the overall data showed that apocytochrome c 1 interacted via its C-terminal helix with CcmI-2 domain, but that it might also interact via its membrane anchor with CcmI-1 domain of CcmI. It remains to be seen whether the second transmembrane helix of CcmI-1 (present in the CcmI-2 variant used here) is also involved in the interactions between the apocytochrome c 1 and CcmI.
How the class I c-type apocytochromes interact with the TPR motifs is not well known. The TPR-containing domain of E. coli NrfG (a functional homologue of R. capsulatus CcmI and E. coli CcmH), which is specific for the maturation of unusual c-type cytochromes (with a heme-binding sequence of C 1 XXC 2 K like the E. coli NrfA (28)) exhibits a K D of ϳ10 M toward a peptide mimicking the C terminus of NrfA. In this case, the TPR binding groove apparently recognizes a helix followed by a loop composed of six C-terminal residues (28). However, the TPR proteins are highly versatile with respect to the amino acid compositions of their TPR motifs, which modulate their ligand affinity. The three-dimensional structures of these proteins bound to their ligands show an intricate network of contacts, including electrostatic, hydrophobic, and Van der Waals interactions (27). For example, such a structure between a TPR protein and the Hsp70 or Hsp90 proteins define a conserved peptide (Met-Glu-Glu-Val-Asp) at the C termini of these proteins binding to the concave groove of the TPR protein (57). Yet, other TPR proteins present alternative interaction modes, including a binding site not located in the TPR groove but composed of the loops connecting the TPR domains (58). Clearly, much remains to be learned about the interactions of the c-type apocytochromes with their TPR-containing chaperones. Reduced minus oxidized optical difference spectra between 380 and 650 nm of 6.5 M b-type cytochrome derivative formed after stoichiometric addition of hemin to 10 M of H. thermophilus apocytochrome b-c 552 (purple), which was used as a "heme reconstitution" control. Similar reconstitution experiments were repeated using the same amounts of R. capsulatus apocytochrome c 2 (black), apocytochrome c 1 (blue), and apocytochrome cЈ (red) with stoichiometric amounts of hemin, and the obtained spectra were compared taking as 100% that of H. thermophilus apocytochrome b-c 552 . Inset, spectra depict the ␤and ␣-bands of the b-type cytochrome derivatives of R. capsulatus apocytochromes c 2 , c 1 t39, and cЈ.
This study also showed that the heme-handling protein apoCcmE interacts not only with apocytochrome c 2 (37) but also with other c-type apocytochromes. However, these interactions are weaker (M versus nM K D values) than those seen with CcmI but are still discriminatory between the different classes of c-type apocytochromes, with about a 100-fold lower binding affinity for the class II apocytochrome cЈ. They are also unaffected by the redox state of the Cys residues at the hemebinding sites of apocytochromes.
Binding of Heme to c-Type Apocytochromes in the Absence of the Ccm Components-Heme triggers the folding of unstructured c-type apocytochromes, increasing their secondary structures (46). Several c-type apocytochromes, like the mitochondrial apocytochrome c (46,59), Paracoccus denitrificans apocytochrome c 550 (59)  Loose CcmI: apocyt c 2 complex formation of thioether bonds FIGURE 9. CcmI-apocytochrome c 2 interactions in the absence and presence of hemin during the Ccm process. A, shows the Ccm machinery and its two substrates, apocytochrome c 2 and heme, forming holocytochrome c 2 . B, depicts the interactions between CcmI and apocytochrome c 2 in the absence and presence of hemin (thought to be provided by CcmE). This hypothetical scheme takes into account all available data to show that the CcmI-2 portion of CcmI binds tightly the C-terminal helix of apocytochrome c 2 , bringing its heme-binding site near CcmE (left panel). Upon the availability of heme via CcmE, apocytochrome c 2 binds heme noncovalently to form a b-type cytochrome intermediate that interacts less tightly with CcmI-2 (middle two panels). The subsequent formation of covalent thioether bonds between heme and apocytochrome c 2 , via currently unknown steps catalyzed by the remaining components of Ccm System I, yields holocytochrome c 2 (right panel). apocytochromes from thermophilic organisms like Hydrogenobacter thermophilus cytochrome c 552 (51), Aquifex aeolicus cytochrome c 555 (60), Thermus thermophilus cytochrome c 552 (61), and H. thermoluteolus cytochrome cЈ (62) exhibit secondary structures with high helical contents in the absence of heme (52,62,63) and readily yield b-type cytochrome derivatives upon heme availability in vitro (64). Even in exceptional cases, thermophilic molten globular c-type apocytochromes yield b-type cytochrome intermediates that are conducive to spontaneous covalent heme ligation (51). Hence, they are poorer models for cytochrome c maturation studies because of their structures that evolved to bind and ligate heme independently of the Ccm System I.
CD spectral data indicated that no direct correlation exists between the increased helical contents of c-type apocytochromes and their ability to yield b-type cytochrome derivatives upon addition of heme. For example, the class I apocytochrome c 1 exhibited a high degree of secondary structure but was less efficient than apocytochrome c 2 for binding heme (Fig.  8). On the other hand, R. capsulatus apocytochrome cЈ had no secondary structure discernable by CD spectroscopy, like its homologue from mesophilic Allochromatium vinosum (62), and was unable to bind heme and yield a b-type cytochrome derivative. Conversely, an apocytochrome c-b 562 derivative of E. coli cytochrome b 562 (structural homologue of cytochrome cЈ) exhibits a fold that closely matches its final conformation and efficiently incorporates a stoichiometric amount of hemin independently of the Ccm machinery. So far, the only known mesophilic c-type apocytochrome that binds heme efficiently to form a b-type cytochrome derivative is horse cytochrome c (46, 59), but it is matured naturally by the cytochrome c biogenesis System III (65). Although a few c-type apocytochromes, because of their structure and intrinsic stability, can bind heme to yield either b-type cytochromes derivatives or mature c-type cytochromes in the absence of the Ccm components, apparently the "designed" four-helical bundles, which bind heme efficiently, still need the Ccm machinery to produce their c-type cytochrome derivatives (66). Thus, the high helical content of a c-type apocytochrome in the absence of hemin is not predictive of a form that is conducive to efficient heme binding. In addition to the interactions between the heme iron and its axial ligands, as well as those between the porphyrin ring and the polypeptide, the amino acid sequences of c-type apocytochromes might also contribute to providing a suitable environment for trapping heme. If this structural information is virtually absent in a c-type apocytochrome, then it relies heavily on the Ccm processes.
Effect of Hemin on the Interactions of Apocytochrome c 2 with CcmI-A remarkable finding of this study is the very high binding affinity (ϳnM) of CcmI for class I c-type apocytochromes (Fig. 9). Clearly, this high affinity is advantageous for the efficient capture of c-type apocytochromes by the Ccm complex following their translocation across the cytoplasmic membrane. However, it also raises an intriguing issue, which is their subsequent release upon maturation. Our earlier observation that CcmI does not bind holocytochrome c 2 suggests that heme, and probably heme-mediated folding, might affect these interactions. Indeed, the addition of hemin to apocytochrome c 2 promoted the formation of its b-type cytochrome-like derivative (Fig. 9). Co-purification assays in the presence of hemin indicated that the amount of CcmI that co-purified with apocytochrome c 2 decreased. Interestingly, the decrease (ϳ25%) in the amount of CcmI that was retained by apocytochrome c 2 coincided roughly with the amount (ϳ20%) of heme incorporated into apocytochrome c 2 to yield its b-type derivative in vitro. This coincidence suggested that the b-type cytochrome derivative formed upon the addition of hemin interacted poorly with CcmI. This suggestion was also supported by decreased conformational changes seen by the CD spectra of CcmI-apocytochrome c 2 complexes in the presence of hemin (Fig. 7). In agreement with these observations, the quantitative binding assays done in the presence of hemin documented higher K D values for the binding of apocytochrome c 2 to CcmI (Table 3). It should be noted that thorough analyses of the tripartite interactions among hemin, apocytochrome c 2 , and CcmI are complicated due to the heterogeneity induced by binding of hemin to only a fraction of apocytochrome c 2 to yield a b-type cytochrome-like derivative in the absence of any Ccm component in vitro. Nonetheless, the data analyses based on a 2:1 heterogeneous kinetic model supported a decrease in the affinity of CcmI for apocytochrome c 2 (and possibly for other class I c-type apocytochromes) in the presence of hemin, possibly by promoting the formation of a partially folded apocytochrome c 2 with coordinated heme moiety.
In summary, the detailed analyses of the binding process of CcmI to apocytochrome c 2 summarized in Fig. 9 support our current notion that the release of a c-type apocytochrome from its chaperone during the Ccm process could occur upon the availability of heme (probably via CcmE). If so, then a b-type cytochrome derivative forms as an intermediate, which subsequently undergoes covalent heme ligation by the remaining components of the Ccm complex.