The roles of Rhodobacter sphaeroides copper chaperones PCu(A)C and Sco (PrrC) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-type cytochrome c oxidases

Abstract

The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa(3)-type CcO containing a di-copper Cu(A) center and mono-copper Cu(B), plus a cbb(3)-type CcO that contains Cu(B) but lacks Cu(A). Three copper chaperones are located in the periplasm of R. sphaeroides, PCu(A)C, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu(B) of the aa(3)-type but not the cbb(3)-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu(A) assembly in mitochondria and bacteria, and with Cu(B) assembly of the cbb(3)-type CcO. PCu(A)C is present in many bacteria, but not mitochondria. PCu(A)C of Thermus thermophilus metallates a Cu(A) center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa(3)- and cbb(3)-type CcOs of R. sphaeroides has been examined in strains lacking PCu(A)C, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu(2+). The absence of PrrC has a greater effect than the absence of PCu(A)C and PCu(A)C appears to function upstream of PrrC. Analysis of purified aa(3)-type CcO shows that PrrC has a greater effect on the assembly of its Cu(A) than does PCu(A)C, and both chaperones have a lesser but significant effect on the assembly of its Cu(B) even though Cox11 is present. Scenarios for the cellular roles of PCu(A)C and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu(A) of the aa(3)-type CcO and to Cu(B) of the cbb(3)-type CcO, while the predominant role of PCu(A)C may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Figure 1

A. The effect of dodecyl maltoside on the TMPD oxidase activity of the aa₃-type CcO and the cbb₃-type CcO in purified cytoplasmic membranes. The activity of the aa₃-type CcO was assayed in membranes isolated from R. sphaeroides CBB3Δ, which lacks the structural genes for the cbb₃-type CcO [77]. The activity of the cbb₃-type CcO was assayed in membranes isolated from R. sphaeroides YZ200, which lacks the coxII-III operon for the aa₃-type CcO [78]. The activities of the intact membranes are set to 100% and the assays were performed as described in Methods. The TMPD oxidase activity of the aa₃-type CcO is lost when detergent disrupts its interaction with membrane-bound cytochrome c_y, but the TMPD oxidase activity of the cbb₃-type CcO is retained. Error is standard deviation. B. A representative O₂ electrode tracing of the assay for the TMPD oxidase activity of the aa₃-type and cbb₃-type CcOs. See Methods and Results for details. Intact, purified cytoplasmic membranes (M) are added to the reaction cell and O₂ consumption is initiated by the addition of ascorbate and TMPD (A/T). The first register measures total CcO activity (aa₃ plus cbb₃). After all O₂ has been consumed, dodecyl maltoside (D) is added to solubilize the membranes and thereby disrupt the interaction of cytochrome c_y with the aa₃-type CcO. After two minutes, soybean phospholipids are added (P) followed by humidified O₂, which restores O₂ consumption activity. In the second register, only the activity of the cbb₃-type CcO is measured.

Figure 1

A. The effect of dodecyl maltoside on the TMPD oxidase activity of the aa₃-type CcO and the cbb₃-type CcO in purified cytoplasmic membranes. The activity of the aa₃-type CcO was assayed in membranes isolated from R. sphaeroides CBB3Δ, which lacks the structural genes for the cbb₃-type CcO [77]. The activity of the cbb₃-type CcO was assayed in membranes isolated from R. sphaeroides YZ200, which lacks the coxII-III operon for the aa₃-type CcO [78]. The activities of the intact membranes are set to 100% and the assays were performed as described in Methods. The TMPD oxidase activity of the aa₃-type CcO is lost when detergent disrupts its interaction with membrane-bound cytochrome c_y, but the TMPD oxidase activity of the cbb₃-type CcO is retained. Error is standard deviation. B. A representative O₂ electrode tracing of the assay for the TMPD oxidase activity of the aa₃-type and cbb₃-type CcOs. See Methods and Results for details. Intact, purified cytoplasmic membranes (M) are added to the reaction cell and O₂ consumption is initiated by the addition of ascorbate and TMPD (A/T). The first register measures total CcO activity (aa₃ plus cbb₃). After all O₂ has been consumed, dodecyl maltoside (D) is added to solubilize the membranes and thereby disrupt the interaction of cytochrome c_y with the aa₃-type CcO. After two minutes, soybean phospholipids are added (P) followed by humidified O₂, which restores O₂ consumption activity. In the second register, only the activity of the cbb₃-type CcO is measured.

Figure 2

The activities of the aa₃-type and cbb₃-type CcOs in cytoplasmic membranes purified from wild-type R. sphaeroides cells and from strains containing different amounts of the copper chaperones PrrC (Sco) and PCu_AC. Assays were performed as described in Methods and Results. A. Oxidase activities in membranes isolated from cells grown in 1.6 µM Cu²⁺ B. Oxidase activities in membranes isolated from cells grown in <50 nM Cu²⁺ Error is standard deviation.

Figure 2

The activities of the aa₃-type and cbb₃-type CcOs in cytoplasmic membranes purified from wild-type R. sphaeroides cells and from strains containing different amounts of the copper chaperones PrrC (Sco) and PCu_AC. Assays were performed as described in Methods and Results. A. Oxidase activities in membranes isolated from cells grown in 1.6 µM Cu²⁺ B. Oxidase activities in membranes isolated from cells grown in <50 nM Cu²⁺ Error is standard deviation.

Figure 3

EPR spectra of purified aa₃-type CcO assembled in wild-type cells grown in 1.6 µM Cu²⁺ or <50 nM Cu²⁺ (low Cu), and in cells lacking PCu_AC or PrrC and grown in <50 nM Cu²⁺. The spectra were recorded at X band using a Bruker (Billerica, MA) EMX spectrometer. Each spectrum is an average of ten scans taken at 10 K using 25–50 µM CcO. The spectra were recorded using a microwave power of 2 mW at 9.38 GHz. The sweep time was 160 s, and the time constant was 83 ms. The amplitudes of the spectra were normalized by the heme a content of the samples as described in Results.

Figure 4

Copper content per CcO of the aa₃-type CcO samples used in Figure 3. The number of coppers per CcO was determined by ICP-OES as described in Methods. Error is standard deviation.

Figure 5

The relative Cu_A content, Cu_B content and O₂ reduction activity of purified aa₃-type CcO assembled in wild-type cells grown in 1.6 µM Cu²⁺ or <50 nM Cu²⁺ (low Cu), and in cells lacking PCu_AC or PrrC and grown in <50 nM Cu²⁺. The fractional content of Cu_A, relative to wild-type cells grown in 1.6 mM Cu²⁺, is taken from the amplitudes of the g = 2.03 signal in Figure 3. The fractional content of Cu_B is estimated as described in Results. The relative activities are taken from the TN_max values for cytochrome c-driven O₂ reduction measured at pH 6.5 as described in Varanasi and Hosler [59]. The TN_max for the wild-type aa₃-type CcO grown in 1.6 mM Cu²⁺ (100%) is 1717 ± 41 e⁻ sec⁻¹.

Figure 6

Possible schemes for the functions of PCu_AC and PrrC in the assembly of Cu_A of the aa₃-type CcO. Explanations are provided in the text. In Scheme A the sulfurs (S) are those of the two cysteine ligands of Cu_A.