Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import

ABSTRACT

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb₃-type cytochrome c oxidase (cbb₃-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met₂₃₃xxxMet₂₃₇ and His₂₆₁xxxMet₂₆₅ motifs in its TMH7 and TMH8, respectively. Of these residues, Met₂₃₃ and His₂₆₁ are essential for Cu uptake and cbb₃-Cox production, whereas Met₂₃₇ and Met₂₆₅ contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met₂₃₇ and Met₂₆₅ completely abolishes Cu uptake and cbb₃-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys₄₉ located at the end of TMH2, the Cys₂₄₇ on a surface loop between TMH7 and THM8, and the C₃₆₇ located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb₃-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function.

INTRODUCTION

The major facilitator superfamily (MFS) is one of the largest groups of secondary active transporters that are exceptionally diverse and ubiquitous to all three kingdoms of life. MFS members selectively transport a wide range of substrates, including sugars, amino acids, peptides, and antibiotics, either by utilizing the electrochemical gradient due to differential substrate concentration or by coupling the transport of one substrate to that of another via the so-called alternating-access mechanism (1, 2).

A typical MFS protein comprises 400 to 600 amino acids often arranged as 12 transmembrane α-helices (TMHs) in two pseudosymmetrical six N-terminal (N-ter) and six C-terminal (C-ter) TMH bundles, with both termini on the inner (n) side of the cytoplasmic membrane. In most cases, MFS proteins contain a substrate-binding cavity enclosed by the N- and C-ter domains and located about halfway into the membrane. The three-dimensional (3D) structures of several MFS transporters are available, and they exhibit different conformational states, supporting an alternating-access cycle mechanism of function (3–5). Accordingly, MFS-type transporters undergo a series of conformational changes to upload, and subsequently to release, their substrates across the membrane. These conformations are referred to as outward-open, outward-facing-occluded, inward-facing-occluded, and inward-open states. The interactions of the substrate with specific residues during its binding and its release are thought to trigger the dynamics of interdomain interactions (3, 6). The nature and location of the residues that selectively bind the substrate and those that trigger the conformational changes required for transport may differ among the MFS proteins. In the case of the lactose permease LacY, the residues TMH4-Glu₁₂₆ and TMH5-Arg₁₄₄ are essential for sugar binding, whereas the TMH7-Tyr₂₃₆, TMH8-Glu₂₆₉, and TMH10-His₃₂₂, which are close to one another in the inward-facing structure, coordinate the closing and the opening of the protein upon substrate binding and release (7, 8). For the oligopeptide transporter PepT, the residues TMH1-Tyr₂₉, TMH1-Tyr₃₀, and TMH2-Tyr₆₈ are important for peptide binding, while TMH10-Gly₄₀₇ and TMH11-Trp₄₂₇ form the pivotal points that control the occluded and the inward-facing conformational states of PepT (9).

The MFS-type transporter CcoA was the first copper (Cu) importer reported in bacteria (10–12) and has become the prototype of the large CalT (CcoA-like Transporters) subfamily of Cu transporters (13). It was initially identified in the facultative phototroph Rhodobacter capsulatus, where it is required for the biogenesis of the binuclear heme-Cu (Cu_B) center of cbb₃-type cytochrome c oxidases (cbb₃-Cox) (14), a C family of heme-Cu O₂ reductase. Comparative phylogenomics of CcoA orthologs showed that they are widespread among the alphaproteobacteria (15). In species like Rhodobacter sphaeroides, which produces multiple heme-Cu O₂ reductases, CcoA is specific to cbb₃-Cox and not involved in the maturation of the closely related aa₃-type Cox (15). This is noteworthy because both the cbb₃-Cox and aa₃-Cox have similar heme-Cu_B centers (16). Distant orthologs of R. capsulatus CcoA, initially thought to transport riboflavin, were shown to import Cu (13), and their occurrence in some proteobacteria lacking cbb₃-Cox suggests that CcoA-imported Cu is likely destined to other cuproproteins in these species. Thus, the CalT family members might have a broader role extending beyond the cbb₃-Cox biogenesis.

Previous studies addressing the Cu binding and import functions of CcoA revealed two motifs, M₂₃₃xxxM₂₃₇ and H₂₆₁xxxM₂₆₅, that are well conserved among its homologs (17). The membrane topology of CcoA and locations of these motifs in the predicted TMH7 and TMH8 suggested that they are parts of its membrane-buried Cu binding site (Fig. 1, left panel). Substitution of these residues with alanine, which is unable to ligand metals, and analyses of the ensuing mutants for ⁶⁴Cu uptake and cbb₃-Cox production had shown that the M₂₃₃ and H₂₆₁ residues are essential for CcoA activity, whereas substitution of M₂₃₇, or M₂₆₅, which are also parts of the conserved motifs, only partially affected the function (17). The putative Cu binding site of CcoA suggested that its mode of action was likely to be different from other well-studied eukaryotic Cu transporters, such as the Ctr-type (18) or the P-type (19) ATPase proteins.

FIG 1.

CcoA homology model using YajR as a template (CcoA_YajR) and locations of its selected Met, His, and Cys residues. Different views of CcoA_YajR model generated using SWISS-MODEL (http://swissmodel.expasy.org; GMQE 0.51) are shown to highlight the respective locations of the conserved motifs (M₂₃₃xxxM₂₃₇ and H₂₆₁xxxM₂₆₅) proposed to bind Cu (left), the selected Met (M₃₀, M₃₂, M₆₉, M₇₃, and Met₂₂₇) and His (H₂₄₉ and H₂₇₄) (middle), and the Cys (C₄₉, C₁₀₉, C₂₂₅, C₂₄₇, and C₃₆₇) (right) residues of CcoA examined in this study. The Met, His, and Cys residues are colored in red, green, and yellow, respectively.

Here, Cu import by R. capsulatus CcoA was studied in further detail. First, to better define the role of the M₂₃₇ and M₂₆₅ residues, a double mutant, M₂₃₇A+M₂₆₅A, was obtained and shown to be completely defective for Cu import and cbb₃-Cox activity, in contrast to the respective single mutants. In addition, the roles of selected five Met (M₃₀, M₃₂, M₆₉, M₇₃, and M₂₂₇), two His (H₂₄₉ and H₂₇₄), and five Cys (C₄₉, C₁₀₉, C₂₂₅, C₂₄₇, and C₃₆₇) residues of CcoA (Fig. 1, middle and right panels) were examined by monitoring the cbb₃-Cox activities after mutagenesis. The results support a hypothetical model for R. capsulatus CcoA function, possibly involving thiol:disulfide exchange reactions between its periplasm-facing Cys residues during Cu import.

RESULTS

Either M₂₃₇ or M₂₆₅ residues of CcoA are required for Cu import.

Earlier studies showed that mutants lacking CcoA were unable to accumulate ⁶⁴Cu in a CcoA-specific (i.e., temperature-dependent) manner (12). Indeed, mutagenesis of the M₂₃₃ and H₂₆₁ residues of CcoA conserved motifs (M₂₃₃XXXM₂₃₇ and H₂₆₁XXXM₂₆₅) (Fig. 1, left panel) completely abolished cbb₃-Cox activity (Table 1), and cellular ⁶⁴Cu accumulation, while mutating M₂₃₇ or M₂₆₅ only partially decreased these activities (17). The results indicated that the conserved M₂₃₃ and H₂₆₁ residues of CcoA are essential for its function, likely forming its intramembrane Cu binding site. However, this study was less informative about the role(s) of the remaining M₂₃₇ and M₂₆₅ residues of the CcoA conserved motifs (17). To further pursue this issue, a double mutant (M₂₃₇A+M₂₆₅A) lacking both of these Met residues was obtained, and its properties were compared to the corresponding single mutants. Both Escherichia coli (Fig. 2A) and R. capsulatus (Fig. 2B) cells harboring the double mutant M₂₃₇A+M₂₆₅A produced CcoA variant proteins at wild-type levels. The direct effects of these mutations on CcoA-dependent Cu uptake were determined by monitoring radioactive ⁶⁴Cu accumulation in whole cells (see Materials and Methods). Both E. coli and R. capsulatus cells producing this CcoA variant were deficient for ⁶⁴Cu uptake (Fig. 3A and B), similar to those mutants lacking CcoA. The cbb₃-Cox activity of the double mutant was also very low (∼2% of wild type), in contrast to ∼73% and ∼35% of the single M₂₃₇A and M₂₆₅A mutants, respectively (Table 1). The R. capsulatus strain lacking a chromosomal copy of ccoA and complemented with a plasmid-borne wild type allele (ΔccoA/plasmid-born ccoA) (see Table S1 in the supplemental material) was used as a control and exhibited a cbb₃-Cox activity of 846 ± 32 μmol of tetramethyl-p-phenylenediamine (TMPD) oxidized/min/mg of total membrane proteins (referred to as 100% in Table 1). Considering that the CcoA variant lacking both M₂₃₇ and M₂₆₅ residues was unable to import Cu and produce active cbb₃-Cox, we concluded that at least one additional Met residue (preferentially M₂₆₅, suggested by its more severe phenotype) located three residues apart from the M₂₃₃ or H₂₆₁ on TMH7 or TMH8, respectively, is required for Cu import, probably as a Cu binding ligand.

TABLE 1.

Various R. capsulatus CcoA mutants and their general properties

Strain	Residue locationb	Conservation (%)c		cbb3-Cox activity
		Rhodobacteriales	Proteobacteria
Residues located in the conserved motifs of CcoAa
ΔccoA/ccoA (wt)*	NAd	NA	NA	100e
SE8 (ΔccoA)*	NA	NA	NA	2
M233A*	TMH7	100	100	9
M237A*	TMH7	100	100	73
H261A*	TMH8	100	100	3
M265A*	TMH8	100	100	35
M237A+M265A	TMH7+THM8	100	100	2
Residues located elsewhere in CcoA
M30A	TMH1	90	10	78
M32A	TMH1	80	0	72
M69A	TMH2	0	0	88
M73A	n side loop	100	60	29
M227A	TMH7	50	20	75
H249A	p side loop	10	40	87
H274A	TMH8	90	30	71
C49A	p end of TMH2	80	0	37
C109A	p end of TMH4	0	70	75
C225A	TMH7	100	20	70
C247A	p side loop	100	50	6
C367A	p end of TMH11	80	0	69

^a*, Values were obtained from Khalfaoui et al. (16, 17) for comparative purposes.

^bp and n refer to the periplasmic and cytoplasmic sides, respectively, of the cytoplasmic membrane.

^cThat is, the percent conservation among the 10 Rhodobacteriales species shown in Fig. S1 and the 10 Proteobacteria species shown in Fig. S2.

^dNA, not applicable.

^e100% cbb₃-Cox activity corresponds to 846 ± 32 μmol of TMPD oxidized/min/mg of total membrane proteins.

FIG 2.

Production of mutant CcoA variants in E. coli and R. capsulatus. Membranes prepared from l-ara induced E. coli (LMG194) (A) and R. capsulatus ΔccoA (SE8) (B) strains harboring appropriate plasmids (see Table S1) expressing wild-type or indicated CcoA mutant variants were probed with anti-Myc monoclonal antibodies. pBAD and CcoA (wt) correspond to membranes prepared from E. coli (A) or R. capsulatus (B) strains harboring empty pBAD (E. coli) or pRK-pBAD (R. capsulatus) expression plasmids, and their derivatives containing Myc-tagged versions of wild-type and mutant ccoA alleles, as appropriate. All native and mutant proteins were produced adequately in both backgrounds.

FIG 3.

Whole-cell radioactive ⁶⁴Cu uptake by CcoA M₂₃₇A+M₂₆₅A double and Cys single mutant derivatives. (A to C) ⁶⁴Cu uptake kinetics observed with the CcoA M₂₃₇A+M₂₆₅A double mutant and C₄₉A, C₁₀₉A, C₂₄₇A, and C₃₆₇A single mutants using appropriate l-ara induced E. coli (LMG194) (A) and R. capsulatus ΔccoA* (ΔccoA ΔcopA, SE24) (B and C) cells, expressing l-ara-inducible native and mutant ccoA alleles. pBAD and ΔccoA refer to control strains, as appropriate. Uptake assays were performed as described in Materials and Methods. Activities measured in cells kept on ice were subtracted from those measured in cells incubated at 37°C, and assays were repeated at least three times using multiple independent cultures. CcoA (wt) refers to (ΔccoA+plasmid-borne ccoA) strain carrying l-ara-inducible native CcoA, where ΔccoA* is ΔccoA ΔcopA (SE24), used to avoid frequent CopA revertants seen with a ccoA deletion (see Table S1) (12). Error bars correspond to the standard deviations around the mean values. In each case, at least three biological and three technical repeats were performed.

TABLE S1

Strains and plasmids. Download Table S1, DOCX file, 0.02 MB ^{(24.6KB, docx)} .

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FIG S1

CcoA of Rhodobacteriales. Download FIG S1, PDF file, 0.1 MB ^{(117.3KB, pdf)} .

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FIG S2

CcoA of Proteobacteria. Download FIG S2, PDF file, 0.1 MB ^{(117.8KB, pdf)} .

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Additional possible metal-liganding CcoA residues of functional importance.

CcoA is rich in Met, His, and Cys residues that can act as potential metal ligands (10), and those that are not parts of the conserved motifs were examined for their possible role(s). Two different amino acid sequence alignments were used with the proteobacterial homologs of CcoA that contributed to building the protein similarity network of the CalT subfamily (13). The R. capsulatus CcoA sequence was first aligned with its closely related CcoA homologs among the Rhodobacterales (mostly from node 1 [see reference 13]) (see Fig. S1). This comparison included R. sphaeroides CcoA known to provide Cu to cbb₃-Cox (16). The R. capsulatus CcoA sequence was also aligned with its more distant homologs among the different proteobacterial orders, including Rhizobiales, Burkhorderiales, Pseudomonales, Rhodospirales, Vibrionales, Oceanospiralles, Synecchocales, Alteromonadales, and Chromatiales (see Fig. S2). This group included Ochrobactrum anthropi (Rhizobiales) CalT (CcoA ortholog) shown to transport Cu (13) and possibly required for the maturation of cuproproteins distinct from cbb₃-Cox. Based on sequence alignments and topological locations (i.e., TMH or loop; Table 1) five Met (M₃₀, M₃₂, M₆₉, M₇₃, and M₂₂₇), possibly forming the M₃₀xM₃₂ and M₆₉xxxM₇₃ motifs (of which M₃₀ and M₇₃ are better conserved), two His (H₂₄₉ and H₂₇₄), and five Cys (C₄₉, C₁₀₉, C₂₂₅, C₂₄₉ and C₃₆₇) residues were retained for this study (Fig. 1, middle and right panels). They were substituted with Ala using l-ara-inducible ccoA, and the mutants obtained were introduced into appropriate E. coli and R. capsulatus strains (see Materials and Methods; Table S1). Their cbb₃-Cox and ⁶⁴Cu uptake activities were determined analogously to the conserved motifs mutants (17).

Properties of CcoA Met and His mutants.

The CcoA single Met (M₃₀A, M₃₂A, M₆₉A, M₇₃A, and M₂₂₇A) mutants were produced in amounts similar to the wild type and M₂₃₇A+M₂₆₅A double mutant in both E. coli and R. capsulatus strains (Fig. 2, since the data are identical for all mutants tested, only M₃₀A and M₃₂A are shown). Of these residues, the M₃₀, M₃₂, M₆₉, and M₇₃ form the so-called “Met (MxM and MxxxM) motifs,” sometimes implicated in binding Cu(I) (20). The single M₃₀A, M₃₂A, and M₆₉A mutants on the N-ter domain and the M₂₂₇A single mutant located close to the M₂₃₃xxxM₂₃₇ putative Cu binding motif on TMH7 had cbb₃-Cox activities slightly lower than that of wild-type CcoA (approximately 78, 72, 88, and 75% of wild-type amounts, respectively), similar to the single mutant M₂₃₇A (∼73%) (Table 1). In contrast, mutating M₇₃ that is located near the cytoplasmic end of TMH2 and highly conserved among the Rhodobacteriales CcoA homologs, led to a markedly lower cbb₃-Cox activity (∼29%) like the single mutant M₂₆₅A (∼35%) (Table 1) (17). The His₂₄₉ and H₂₇₄ are near the periplasmic and cytoplasmic (p and n) sides of the membrane, respectively, and only the latter residue is conserved in Rhodobacteriales (see Fig. S1). The corresponding mutants had cbb₃-Cox activities slightly lower than the wild type (ca. 87 and 71%, respectively) (Table 1). Thus, unlike M₂₃₃ and H₂₆₁, none of these Met and His residues were required for CcoA function, except M₇₃ located close to the cytoplasm, as its substitution significantly reduces cbb₃-Cox activity.

Properties of CcoA Cys mutants.

Of the five Cys residues of CcoA, four (except C₁₀₉) are well-conserved especially among the Rhodobacteriales (see Fig. S1). Based on the homology model of CcoA (CcoA_YajR) obtained using its highest homolog E. coli YajR (PDB 3WDO) as a template, the C₄₉ and C₃₆₇ residues are at or near the periplasmic ends of the TMH2 and TMH11, respectively, while C₂₄₇ is located on a periplasmic loop between TMH7 and TMH8 (Fig. 1, right panel) (10, 17). The nonconserved C₁₀₉ on TMH3, and the partly conserved C₂₂₅ on TMH7 are more deeply embedded into the membrane, closer to the p and n sides, respectively. Substitution of each of these Cys residues with Ala did not affect the production of mutant proteins either in E. coli or R. capsulatus (Fig. 2; only C₄₉, C₁₀₉, and C₂₄₇ are shown). However, it impaired the cbb₃-Cox activity of the mutant strains to different extents (Table 1). While the effects of C₁₀₉A, C₂₂₅A, and C₃₆₇A mutations were milder (ca. 75, 70, and ∼69% of wild-type activity, respectively), those of the C₄₉A and C₂₄₇A were more severe (ca. 37 and ∼6%, respectively). In respect to Cu import, upon l-arabinose (l-ara) addition radioactive ⁶⁴Cu accumulation in whole cells of a control strain lacking CcoA but harboring a plasmid-borne inducible CcoA (ΔccoA+plasmid-borne ccoA) steadily increased, unlike the ΔccoA strain lacking CcoA. With the C₄₉A or C₂₄₇A mutants, ⁶⁴Cu accumulation was very low, similar to the strain lacking CcoA, indicating that these residues are critical for CcoA function (Fig. 3C). On the other hand, the C₁₀₉A and C₃₆₇A mutants accumulated ⁶⁴Cu markedly more slowly to a slightly lower level than the control cells, indicating that C₁₀₉ and C₃₆₇ also contribute to Cu uptake but are not essential. Overall, the data indicated that these mutants fall into two groups (C₄₉A and C₂₄₇A versus C₁₀₉A and C₃₆₇A) with distinct kinetics behaviors, suggesting likely different functions.

Topological locations of periplasm-facing Cys residues of CcoA.

Currently, no 3D structure of CcoA is available beyond the CcoA_YajR homology model (Global Model Quality Estimate [GMQE] 0.51) based on its most pronounced homolog, which is the E. coli YajR (PDB 3WDO) (17). Fortunately, additional homology models of CcoA of similar GMQE yielding similar outcomes can be generated using available X-ray structures, including the iron exporter BpFPN (21). Here, we opted for two of its importer homologs, LacY (PDB 6C9W, CcoA_LacY, GMQE 0.46) and GlpT (PDB 1PW4, CcoA_GlpT, GMQE 0.44), captured in different conformations than CcoA_YajR. While an outward-open conformation (i.e., ready to receive the substrate from the p side of the membrane) is seen with CcoA_YajR, the CcoA_LacY and CcoA_GlpT models provide the occluded and the inward-open (i.e., ready to release the substrate to the n side of the membrane) conformations, respectively (Fig. 4A). Top views of these models clearly show that the distances separating the periplasm facing Cys residues change drastically depending on the conformations (Fig. 4B; see also Table S3, which lists all appropriate α-C-to-α-C distances). When CcoA_YajR is in the outward-open conformation (Fig. 4B top), C₄₉ and C₁₀₉ located on the N-ter domain are very close to each other (C₄₉-C₁₀₉, 12 Å) and distant from C₂₄₇ (C₄₉-C₂₄₇, 32 Å; C₁₀₉-C₂₄₇, 39 Å) and C₃₆₇ (C₄₉-C₃₆₇, 22 Å; C₁₀₉-C₃₆₇, 32 Å) located on the C-ter domain. In the occluded conformation of CcoA_LacY (Fig. 4B, middle), C₄₉ moves closer to C₂₄₇ (C₄₉-C₂₄₇, 23 Å) and C₃₆₇ (C₄₉-C₃₆₇, 16 Å), while C₁₀₉ shifts closer to both C₂₄₇ and C₃₆₇ (C₁₀₉-C₂₄₇, 36 Å; C₁₀₉-C₃₆₇, 28 Å). In the inward-open conformation of CcoA_GlpT (Fig. 4B, bottom), C₄₉ and C₁₀₉ approach even closer to C₂₄₇ (C₄₉-C₂₄₇, 16 Å; C₁₀₉-C₂₄₇, 27 Å) and C₃₆₇ (C₄₉-C₃₆₇, 10 Å; C₁₀₉-C₃₆₇, 22 Å). In all conformations, the N-ter-located C₄₉-C₁₀₉ pair stays within 12 to 14 Å, and the C-ter-located C₂₄₇-C₃₆₇ pair remains within 19 to 22 Å of each other. Indeed, these distance estimations are approximations in the absence of 3D structures. Nonetheless, they depict the progressive movement of the N-ter domain C₄₉ toward the C-ter domain C₂₄₇-C₃₆₇ pair during the transition from the outward-open to the inward-open conformations. This observation enticed us to inquire whether the predicted distance changes are related to the Cys residues that are exposed to the oxidizing periplasm undergoing thiol:disulfide oxidoreduction during CcoA function.

FIG 4.

Different CcoA conformations and the distances that separate the periplasmic Cys residues in each conformational state. (A) Side views of R. capsulatus CcoA homology models (CcoA_YajR, CcoA_LacY, and CcoA_GlpT) representing its different conformations using as the templates E. coli YaJR (3WDO) in the outward-facing state (16% identity; GMQE 0.51), lactose permease LacY (1PV6) in the occluded state (12.7% identity; GMQE 0.41), and GlpT (1PW4) in the inward-facing state (11.7% identity; GMQE 0.46) (http://swissmodel.expasy.org). (B) Top views of the homology models shown in panel A, with the distances separating the periplasm-facing Cys residues (yellow balls) in different conformations of CcoA.

TABLE S3

Approximate distances separating the Cys residues αC-αC of CcoA. Download Table S3, DOCX file, 0.01 MB ^{(15.5KB, docx)} .

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Disulfide bonds formed between the Cys residues of CcoA.

The occurrence in vivo of disulfide bond(s) in CcoA was probed using E. coli cells expressing native CcoA or its Cys mutant variants and the thiol-reactive alkylating agent monomethyl-(PEG)24-maleimide (mPEG) (22). Alkylation of free Cys thiols of CcoA by mPEG is expected to increase its molecular weight (MW) by ∼1.2 kDa per free thiol. In the case of disulfide bonds, alkylation occurs only after reduction by dithiothreitol (DTT), and then mPEG further increases the MW by ∼2.4 kDa per reduced disulfide bond. The relative MW changes (M_r) in native and Cys mutant variants of CcoA were followed by SDS-PAGE/immunodetection (Fig. 5 and 6).

FIG 5.

In vivo free thiols and disulfide bonds in native CcoA and its single Cys mutant derivatives. SDS-PAGE, followed by immunoblot analysis of E. coli cells expressing native and single C₄₉A, C₁₀₉A, C₂₂₅A, C₂₄₇A, and C₃₆₇A mutant variants of CcoA. Cells growing in the presence of l-ara were treated mid-log phase for 10 min with or without DTT, followed by TCA precipitation and subsequent alkylation with or without mPEG. The presence of unmodified or mPEG alkylated CcoA was detected using anti-Myc monoclonal antibody and horseradish peroxidase conjugated anti-mouse IgG (see Materials and Methods). The total protein amounts of SDS-solubilized TCA-precipitated pellets could not be determined precisely, leading to variations of protein amounts loaded per lane. Thus, only the qualitive occurrence of M_r shifts in the absence or presence of DTT or mPEG were taken into consideration in this experiment.

FIG 6.

In vivo free thiols and disulfide bonds formed in native CcoA and its double Cys mutant derivatives. The experiments were conducted as described in Fig. 5, except that E. coli cells expressing the native (left side panels) and double mutant variants of CcoA were used: C₄₉A-C₂₄₇A (A), C₂₄₇A-C₃₆₇A (B), C₄₉A-C₁₀₉A (C), C₁₀₉A-C₃₆₇A (D), and C₁₀₉A-C₂₄₇A (E). As in Fig. 5, the total protein amounts of SDS-solubilized TCA-precipitated pellets could not be determined precisely, leading to variations of protein amounts loaded per lane. Thus, only the M_r shifts detected in the absence or presence of DTT or mPEG were considered in these experiments.

Under our conditions, native CcoA (predicted MW of 37.4 kDa) runs as a band of ∼35-kDa M_r in its oxidized or reduced forms (Fig. 5, left panels), which is not uncommon for membrane proteins. In the absence of DTT, alkylating by mPEG increased native CcoA M_r by ca. 3 to 4 kDa to ca. 38 to 39 kDa, suggesting that it contained three free Cys thiols (predicted M_r of 38.6 kDa after three mPEG addition). Moreover, alkylating native CcoA after DTT reduction further increased its M_r by another ∼2-3 kDa to ca. 40 to 41 kDa, indicating that the native protein contained one disulfide bound in vivo (Fig. 5, left panels). Although detecting the ca. 1- to 2-kDa M_r differences with hydrophobic membrane proteins was challenging, following TCA precipitation all CcoA Cys residues appeared accessible to alkylation, including the Cys₁₀₉ and Cys₂₂₅, which are more buried into the lipid bilayer according to the CcoA structural models (Fig. 1 and 4).

Similar mPEG alkylation experiments were repeated using single Cys mutant variants of CcoA (Fig. 5, right panels). Without DTT reduction, all single Cys mutant variants exhibited mPEG-induced M_r shifts similar to native CcoA, and the largest shift was seen with C₃₆₇A mutant. In all cases but C₃₆₇A, the shifts were consistent with the likely presence of at least two free thiols, but not four as would have been expected upon elimination of any Cys residues already engaged in a disulfide bond in native CcoA. After DTT reduction, all single Cys mutants, except C₃₆₇A, showed the additional mPEG-induced M_r shifts, indicating that they still contained a disulfide bond formed among the remaining Cys residues. This observation suggested that native CcoA has more than two Cys residues that could form a disulfide bond(s). The C₃₆₇A mutant was intensely alkylated but did not exhibit any readily detected mPEG-mediated M_r shift after DTT reduction, indicating that it contained no more disulfide bonds, and suggested that this residue provides one of the thiol groups forming a stable disulfide bond in native CcoA (Fig. 5, right panel, last row). Although occasionally additional minor bands were also seen in some cases (e.g., native CcoA, Fig. 5 left panel, second row, or C₂₂₅A, Fig. 5 right panel, third row), the data showed that the C₄₉A, C₁₀₉A, C₂₂₅A, or C₂₄₇A single Cys mutants behaved similarly to each other and to native CcoA, which precluded identification of partner cysteines for forming a disulfide bond.

To identify the disulfide bond forming partner(s) in native CcoA in vivo, a set of double Cys mutants were examined (Fig. 6). All CcoA double Cys variants were produced adequately in E. coli and in R. capsulatus and exhibited low cbb₃-Cox activities like their cognate single Cys mutants. In the absence of DTT, mPEG alkylation data showed that the double mutants C₄₉A-C₂₄₇A (with C₁₀₉, C₂₂₅, and C₃₆₇ intact) and C₂₄₇A-C₃₆₇A (with C₄₉, C₁₀₉, and C₂₂₅ intact) had M_r shifts similar to each other, and to native CcoA, containing free thiols (Fig. 6A and B). However, like the C₃₆₇ single mutant (Fig. 5, bottom row), these two double Cys mutants did not exhibit any additional mPEG-induced M_r increase upon reduction by DTT, indicating that the remaining Cys residues did not form disulfide bonds. Conversely, the double mutants C₄₉-C₁₀₉ (with C₂₂₅, C₂₄₇, and C₃₆₇ intact) and C₁₀₉-C₃₆₇ (with C₄₉, C₂₂₅, and C₂₄₇ intact) showed no or slight mPEG-induced M_r shifts in the absence of DTT (although the absence of this shift was less clear in the latter double mutant) but exhibited clearer M_r shifts upon mPEG alkylation after DTT treatment (Fig. 6C and D). Since the C₂₂₅ residue is near the n side and remote from the other periplasm-exposed Cys residues on the p side of the membrane, it is likely that in the C₄₉A-C₁₀₉A and C₁₀₉A-C₃₆₇A double mutants, the C₂₄₇ and C₃₆₇ and the C₄₉ and C₂₄₇ residues, respectively, formed disulfide bonds (although the latter pair might form a less stable disulfide bond) (Fig. 6C and D, far right). The slight M_r shifts seen with these double mutants in the absence of DTT reduction were consistent with the poor alkylation of C₂₂₅, still intact in these mutants. The remaining C₁₀₉A-C₂₄₇A double mutant (with C₂₂₅, C₄₉, and C₃₆₇ intact) behaved essentially like the latter mutants, except that the CcoA population appeared heterogenous in the absence of DTT (Fig. 6E). A small fraction contained free thiols that was alkylated by mPEG without DTT treatment, whereas a large fraction contained a disulfide bond that could only be alkylated after DTT reduction. Again, assuming that C₂₂₅ is too far from the other Cys residues to participate in disulfide bond formation, a large fraction of the C₁₀₉A-C₂₄₇A double-mutant population comprises a less stable disulfide bond between the C₄₉ and C₃₆₇ residues (Fig. 6E). In summary, the overall data indicated the formation of disulfide bonds between C₄₉∼C₂₄₇, C₄₉∼C₃₆₇, and C₂₄₇∼C₃₆₇ (with the last one forming the most stable bond) and the clear absence of disulfide bonds between C₁₀₉∼C₃₆₇ and C₄₉∼C₁₀₉. This suggests that Cys₁₀₉ is redox inactive, unlike the other periplasm-exposed residues, possibly due to its membrane-buried location in all conformations of CcoA (Fig. 4). Consequently, in cells producing native CcoA, any pair among the C₄₉, C₂₄₇, and C₃₆₇ residues could form a single disulfide bond in vivo, leaving behind three free thiol groups, including C₁₀₉ and C₂₂₅. This finding raised the possibility that native CcoA in vivo might exist as a heterogenous population with different conformations, presumably due to the import of spurious Cu presumably present in the growth medium. How the initial binding of Cu changes the conformation of CcoA and shuffles the free thiols and disulfide bonds between its three active Cys residues remains to be determined in future studies.

DISCUSSION

This study focused on the role of plausible metal-liganding residues Met, His and Cys of CcoA, a member of the CalT (CcoA-like Transporters) subfamily of MFS-type transporters (13) and the prototype of proteobacterial Cu importers (10, 15). The CalT subfamily is characterized by two well-conserved motifs (M₂₃₃xxxM₂₃₇ and H₂₆₁xxxM₂₆₅ in R. capsulatus CcoA) of which the first Met and His residues are absolutely required for Cu import (17). Here, we show that mutating concomitantly the M₂₃₇ and M₂₆₅ residues also abolishes CcoA activity, unlike the corresponding single mutants. Thus, the presence of at least one additional Met residue together with Met₂₃₃ and His₂₆₁ is required for Cu import. This finding further supports the Cu binding role of the conserved motifs that are the hallmark of the CalT subfamily of MFS-type transporters (13, 17).

We examined the distribution and topological location of additional possible metal-liganding residues of CcoA that are often conserved among its homologs, in particular those from the Rhodobacterales within the Proteobacteria. Of these residues, mutating C₂₂₅, M₂₂₇, or H₂₇₄ located at the TM7 and TM8 on the C-ter domain of CcoA near the membrane Cu-binding site, had little effect on CcoA activity. This finding was similar to that seen with the M₂₃₇A or M₂₆₅A single mutants, suggesting that they were either not critical for function, or partly substituted by surrogate residues. Intriguingly, mutating M₇₃, but not M₆₉, of the putative “Met” motif (M₆₉xxxM₇₃ in R. capsulatus) (20) had a stronger effect on cbb₃-Cox activity. Homology models of the different conformations of CcoA do not seem to suggest that these N-ter residues come very close to the C-ter Cu binding residues. However, how Cu is released from CcoA is not yet known, leaving the possibility open that the C₂₂₅, M₂₂₇, and H₂₇₄ residues or the putative Met (M₆₉xxxM₇₃) motif, or both, all positioned closer to the n side of the membrane, might play a role in this process.

Remarkably, mutating the periplasm-exposed C₄₉, C₂₄₇, and C₃₆₇ residues affected CcoA activity to different degrees. These residues are well-conserved among the Rhodobacterales, but either less (∼50% for C₂₄₇) or not (0% for C₄₉ and C₃₆₇) conserved in other proteobacterial orders (see Fig. S1 and S2). The basis of this conservation is not obvious, but it might relate to the ultimate destination of Cu (e.g., cbb₃-Cox in R. capsulatus and other cuproenzymes in O. anthropi) and the different Cu donors and acceptors of CcoA and its homologs. Of the periplasm-facing Cys (C₄₉, C₁₀₉, C₂₄₇, and C₃₆₇) residues of CcoA, mutating C₁₀₉ slowed Cu uptake (Fig. 3C), slightly affected cbb₃-Cox activity (Table 1), and mPEG alkylation indicated that C₁₀₉ does not form a disulfide bond with either C₃₆₇ or C₄₉. Intriguingly, C₁₀₉ residue is not conserved among the Rhodobacterales (0%) but is better conserved (∼70%) among the other orders of proteobacteria where CalT is thought to provide Cu to other cuproproteins distinct from the cbb₃-Cox (13).

Alkylation data of the single and double Cys mutants revealed that in native CcoA, two of the three periplasm-facing C₄₉, C₂₄₇, and C₃₆₇ residues form a disulfide bond, while the remaining two remain as free thiol in vivo. Moreover, all possible disulfide and free thiol combinations among these residues (i.e., C₂₄₇∼C₃₆₇ leaving C₄₉ free, C₄₉∼C₂₄₇ leaving C₃₆₇ free, and C₄₉∼C₃₆₇ leaving C₂₄₇ free) were observed in appropriate Cys double mutants. However, the levels of stability of these bonds seem to be different, with the C₂₄₇∼C₃₆₇ bond being most stable. Although the data in Fig. 6 tend to suggest that the C₄₉∼C₂₄₇ bond might be formed, yet the data with the single C₃₆₇A mutant (Fig. 5) suggest that it certainly must not be stable to be readily detected in this mutant. Assuming that CcoA undergoes conformational changes like any MFS-type transporter, the disulfide bond formation patterns suggest a hypothetical model linking Cu binding and conformational changes (Fig. 7). Accordingly, in the outward-open conformation of CcoA (state 1), C₂₄₇ and C₃₆₇ would contain a disulfide bond, far away from C₄₉. Binding of Cu would convert CcoA into its occluded conformation (state 2), bringing C₄₉ near the C₂₄₇∼C₃₆₇ disulfide bond, and a nucleophilic attack would yield either C₄₉∼C₃₆₇ (Fig. 7, left) or C₄₉∼C₂₄₇ (Fig. 7, right) disulfide bond while freeing the remaining thiol of C₂₄₇ or C₃₆₇. We note that if no such disulfide bond is formed or is extremely unstable, then the occluded conformation (state 2) may not have a disulfide bond (not shown in Fig. 7). In the exponentially growing cells used in this study, Cu import is not synchronized; thus, different conformations of CcoA must coexist, rendering impossible to discriminate between these possibilities at this stage. The more defective phenotype and the periplasmic location (i.e., increased solvent exposure) of C₂₄₇ as well as the weaker nature of C₄₉∼C₂₄₇ (as suggested by its absence in C₃₆₇A single mutant) and the detection of C₄₉∼C₃₆₇ (as seen with C₁₀₉A-C₂₄₇A double mutant) disulfide bonds might argue that the C₄₉-C₃₆₇ disulfide bond may be more favorable at the inward open conformation (state 3) (Fig. 7, right). In any case, further progression of Cu within CcoA from the periplasm toward the cytoplasm would trigger the remaining free thiol (C₂₄₇ or C₃₆₇) to attack the disulfide bond involving C₄₉ (C₄₉∼C₂₄₇ or C₄₉∼C₃₆₇) at the inward open conformation (state 3). The subsequent resolution of this bond would then reestablish the initial C₂₄₇∼C₃₆₇ disulfide bond and free C₄₉ thiol, returning CcoA to its outward-open conformation (state 1). This model attributing more critical roles to C₄₉ and C₂₄₇ is also consistent with the highly defective ⁶⁴Cu uptake seen with the C₄₉A and C₂₄₇A single mutants (Fig. 3C). Conceivably, the three periplasm-facing C₄₉, C₂₄₇, and C₃₆₇ residues that are highly conserved in Rhodobacterales may also play additional and perhaps different roles (e.g., liganding Cu) instead of those ascribed here. However, this hypothetical model suggests a link between the binding of Cu, ensuing conformation changes, and plausible thiol:disulfide oxidoreduction of CcoA. In this respect, the absence of R. capsulatus thiol: disulfide oxidoreductase DsbA (23), which catalyzes intramolecular disulfide bonds in extracytoplasmic proteins, is known to affect cbb₃-Cox biogenesis (24). Whether or not DsbA is involved in these thiol:disulfide exchange reactions seen with CcoA is presently unknown, but future studies addressing determination of the thiol:disulfide exchange reaction rates (e.g., using 5,5-dithio-bis-2-nitrobenzoic acid) (23) and the pKa values of appropriate thiols might identify the attacking and resolving Cys residues to further elucidate this process.

FIG 7.

Hypothetical model linking substrate binding, conformational changes, and thiol:disulfide exchange reactions that CcoA might undergo during Cu import. Accordingly, at the onset CcoA has one disulfide C₂₄₇∼C₃₆₇ and C₄₉-SH (outward open state 1), binding of Cu induces a conformational change leading to occluded state 2, where a C₄₉∼C₃₆₇ (left side) or a C₄₉∼C₂₄₇ (right side) disulfide bond is formed (although the available data cannot rule out the absence of a disulfide bond at this state). Exchange of disulfide bonds and progress of Cu from the p to the n side of the membrane yield the inward open state 3. Upon release of Cu and resolution of the latter disulfide bond, CcoA is returned to its starting conformation.

Noticeably, other Cu transporters also have critical Cys residues. The Ctr-type transporters contain important Cys residues (within the HCH motif) at their C-terminal parts, near the n side of the membrane (25). They form disulfide bonds between the monomers of trimeric CTR1 to facilitate Cu release, unlike the periplasm-facing Cys residues of CcoA monomer, presumably involved in Cu uptake. Clearly, a requirement for Met, His, and Cys residues for CcoA activity distinguishes its mechanism of function from other well-known Cu(I) transporters, including the eukaryotic Ctr1 (25–27) or the bacterial CopA and CusA (28, 29) proteins.

In summary, we showed here that the periplasm-facing Cys residues, together with the conserved membrane-embedded Cu-binding (M₂₃₃xxxM₂₃₇) and (H₂₆₁xxxM₂₆₅) motifs, are critical for proper function of the MFS-type Cu importer CcoA and possibly of its close homologs among Rhodobacterales.

MATERIALS AND METHODS

Growth conditions, strains, and plasmids used.

The bacterial strains used in this work are described in Table S1 in the supplemental material. Escherichia coli strains were grown at 37°C on LB (lysogeny broth or Luria-Bertani) medium, supplemented with antibiotics (100 and 12.5 μg/ml ampicillin [Amp] and tetracycline [Tet], respectively) and l-arabinose (l-ara; 0.5%), as appropriate (17). The purple nonsulfur facultative photosynthetic R. capsulatus strains were grown at 35°C under respiratory (aerobic dark) conditions in enriched MPYE (minerals, bactopeptone, and yeast extract) medium supplemented with antibiotics (2.5 μg/ml Tet) and l-ara (0.5%), as needed (30).

Construction of CcoA mutants.

Standard molecular genetic techniques were performed as described previously (31). The plasmids and primer sequences used are listed in Tables S1 and S2, respectively. The plasmid pBK68 carrying the l-ara-inducible ccoA is a derivative of pBAD/Myc-HisA (17) and used as a template for site-directed mutagenesis. Appropriate forward and reverse mutagenesis primers (see Table S2) were used to generate the plasmids pBK98 (M₃₀A), pBK99 (M₃₂A), pBK100 (M₆₉A), pBK101 (M₇₃A), pSP6 (M₂₂₇A), pSP4 (H₂₄₉A), pSP5 (H₂₇₄A), pBK108 (C₄₉A), pBK109 (C₁₀₉A), pSP9 (C₂₂₅A), pSP8 (C₂₄₇A), and pBK117 (C₃₆₇A), producing CcoA variants with the indicated mutations (see Table S1). These plasmids served as the templates for generating the ccoA double Met or Cys mutants by using appropriate forward and reverse primers (see Table S2) to yield pBK79 (M₂₃₇A+M₂₆₅A), pBK119 (C₄₉A+C₁₀₉A), pBK122 (C₄₉A+C₂₄₇A), pBK126 (C₁₀₉A+C₂₄₇A), pBK123 (C₂₄₇A+C₃₆₇A), and pBK129 (C₁₀₉A+C₃₆₇A), producing mutant CcoA variants (see Table S1). The pBAD derivatives carrying the mutant alleles of ccoA were cut with NsiI and ligated to the PstI site of the broad-host-range plasmid pRK415 to generate the following pBAD-pRK415 composite plasmid derivatives carrying the mutant ccoA alleles: pBK90 (H₂₄₉A), pBK95 (M₂₃₇A+M₂₆₅A), pBK102 (M₃₀A), pBK103 (M₃₂A), pBK104 (M₆₉A), pBK105 (M₇₃A), pBK92 (M₂₂₇A), pBK91 (H₂₄₉A), pBK111 (C₄₉A), pBK112 (C₁₀₉A), pBK94 (C₂₂₅A), pBK93 (C₂₄₇A), pBK120 (C₃₆₇A), pBK121 (C₄₉A+C₁₀₉A), pBK124 (C₄₉A+C₂₄₇A), pBK127 (C₁₀₉A+C₂₄₇A), pBK125 (C₂₄₇A+C₃₆₇A), and pBK130 (C₁₀₉A+C₃₆₇A) (see Table S1). These pRK derivatives were conjugated into appropriate R. capsulatus strains, as described earlier (17).

TABLE S2

Primers used in this study. Download Table S2, DOCX file, 0.01 MB ^{(15.4KB, docx)} .

Copyright © 2021 Khalfaoui-Hassani et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Chromatophore membrane preparation, SDS-PAGE, and immunodetection.

Intracytoplasmic membrane vesicles (chromatophore membranes) were prepared in 20 mM Tris-HCl (pH 7.0) containing 150 mM NaCl and 1 mM phenylmethylsulfonylfluoride (PMSF) as previously described (30). Total protein concentrations were determined using the bicinchoninic acid assay (Sigma, Inc., procedure TPRO-562), and SDS-PAGE (12%) analyses were conducted as described previously (32). Prior to loading, 20 or 40 μg of proteins samples of E. coli or R. capsulatus, respectively, were solubilized by incubation at room temperature for 10 min in a loading buffer (final concentration of 62.5 mM Tris-HCl [pH 6.8], 2% SDS, 2% β-mercaptoethanol, and 0.01% bromophenol blue). After electrophoresis, gels were electroblotted onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Inc.), and probed with anti c-Myc tag monoclonal antibodies (clone 9E10 from the University of Pennsylvania Cell Center). The secondary antibodies were horseradish peroxidase-conjugated anti-mouse IgGs (GE Healthcare, Inc.), and the signal was detected using Supersignal West Pico chemiluminescence substrate according to the supplier’s recommendations (Thermo Fisher, Inc.).

Determination of the free thiol groups and disulfide bonds of CcoA in vivo.

E. coli cells producing wild-type or appropriate Cys mutant derivatives of CcoA were analyzed by alkylating free Cys thiols with methoxy-polyethylene glycol-maleimide (mPEG-MAL) before and after DTT reduction, as previously described (22). Strains producing desired CcoA variants were grown overnight at 37°C in 5 ml of LB medium supplemented with appropriate antibiotics (Amp, 100 μg/ml; Tet, 10 μg/ml) with shaking at 180 rpm. The next day,100 to 200 μl of these cultures were subcultured into 10 ml of fresh LB medium containing 1% l-ara and appropriate antibiotics at 37°C with shaking (180 rpm) until they reached an optical density at 600 nm (OD₆₀₀) of 0.5. At this stage, two aliquots of 0.9 ml each were taken out and kept on ice, while the remaining culture (8.2 ml) was reduced by addition of 82 μl of 1.0 M DTT (10 mM final concentration) and further incubated for 10 min at 37°C with shaking. Two additional aliquots of 0.9 ml each were taken and placed on ice. All four samples were precipitated by addition of 100 μl of 100% ice-cold TCA (final concentration, 10% [vol/vol]) and incubated on ice for 30 min. Precipitated materials were centrifuged at 13,000 rpm at 4°C for 12 min, and supernatants were removed without disturbing the pellets, which were washed with 300 μl of ice-cold acetone to eliminate TCA, and recentrifuged under the same conditions. The pellets were partially dried at 30°C for ∼10 min to evaporate acetone, one untreated pellet and one DTT-treated pellet were resuspended in 30 μl of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2.0 mM KH₂PO₄ [pH 7.0]) supplemented with 0.1% SDS. Similarly, the remaining one untreated and one DTT-treated pellets were resuspended in 30 μl of mPEG-MAL solution (20 mM mPEG-MAL dissolved in PBS buffer) supplemented with 0.1% SDS. The pellets were vortexed vigorously for 1 min for complete dissolution, followed by incubation in the dark at room temperature for 2 h under constant shaking (1,000 rpm) to label the accessible thiol groups of CcoA. At the end of the incubation, 10 μl of 5× Laemmli buffer (10% SDS [vol/vol], 0.05% bromophenol blue [wt/vol], 60% glycerol [vol/vol], 300 mM Tris-HCl [pH 6.8]) was added to each sample, followed by further incubation at room temperature for 15 min. Then, 20 μl of each sample was loaded on a 12% nonreducing SDS-PAGE gel, run at 200 V, and subjected to immunoblot analyses using α-myc monoclonal antibodies (1:5,000 dilution) as primary antibodies and horseradish peroxidase conjugated anti-mouse IgGs as secondary antibodies (1:3,000 dilution). The addition of mPEG-MAL, specific to free thiol groups, increases the M_r of alkylated mPEG-MAL derivatives of CcoA, with the increases being proportional to the number of free thiol groups. Comparison of untreated and DTT-treated samples prior to mPEG-MAL alkylation visualized the DTT-reduced disulfide bonds of CcoA in vivo under the growth conditions used.

cbb₃-Cox activity.

The in situ cbb₃-Cox activity of R. capsulatus colonies was assessed qualitatively using the “NADI” staining solution, which is made by mixing in a 1:1 (vol/vol) ratio 35 mM α-naphthol and 30 mM N,N,N′,N′-dimethyl-p-phenylene diamine (DMPD) dissolved in ethanol and water, respectively (33). Colonies producing cbb₃-Cox stain blue, while those lacking it remain unstained. The in vitro cbb₃-Cox activity was measured quantitatively using R. capsulatus chromatophore membranes and TMPD by monitoring spectrophotometrically in a stirred cuvette its oxidized form at 562 nm (λ₅₆₂ = 11.7) at room temperature. Briefly, 10 μg of R. capsulatus chromatophore membranes was added to 1 ml of assay buffer (25 mM Tris-HCl [pH 7.0], 150 mM NaCl), and the enzymatic reaction was initiated by addition of TMPD at a final concentration of 1 mM. The TMPD oxidation specifically due to cbb₃-Cox activity was controlled by incubating the chromatophore membranes with 200 μM KCN for 2 min prior to TMPD addition. The cbb₃-Cox activity was calculated by subtracting from the TMPD oxidase activity the fraction that was KCN insensitive (15).

Radioactive ⁶⁴Cu uptake assays.

Cellular Cu uptake assays were performed as previously described (12), using whole cells and radioactive ⁶⁴Cu (1.84 × 10⁴ mCi/μmol specific activity) obtained from Mallinckrodt Institute of Radiology, Washington University Medical School. The. E. coli strains harboring appropriate pBAD/Myc-His derivatives with l-ara-inducible ccoA wild- type and mutant variants (see Table S1) were grown overnight in 10 ml of LB medium supplemented with 0.5% l-ara and appropriate antibiotics. Cells were pelleted, washed with the assay buffer (50 mM sodium citrate [pH 6.5], 5% glucose), and resuspended in 1 ml of the same buffer. All cultures were normalized to the same number of cells (7.5 × 10⁸ cells/500 μl of assay volume) based on their absorbance (1 OD₆₀₀ = 5 × 10⁸ cells/ml for E. coli and 1 OD₆₃₀ = 7.5 × 10⁸ cells/ml for R. capsulatus strains). Cells to be assayed for ⁶⁴Cu uptake were preincubated at 35 or 0°C for 10 min before the assay. The uptake activity was initiated by addition of 10⁷ cpm of ⁶⁴Cu, determined immediately before use (half-life of ⁶⁴Cu isotope ∼12 h), and 50-μl aliquots were taken at 0, 1, 2, 5, and 10 min of incubation and immediately mixed with ice-cold 50 μl of 1 mM CuCl₂ and 50 μl of 50 mM EDTA (pH 6.5) to stop the uptake reaction. All aliquots were kept on ice until the end of the assay; the cells were then pelleted, and the pellets were washed twice with 100 μl of ice-cold 50 mM EDTA solution, resuspended in 1 ml of scintillation liquid, and counted using a scintillation counter (Coulter-Beckman, Inc.) with a wide-open window. For each time point, the background ⁶⁴Cu uptake activity seen at 0°C was subtracted from that at 35°C and plotted as a function of time to compare CcoA-specific Cu uptake of wild-type control (ΔccoA/plasmid-born ccoA) and mutant derivatives of CcoA.