The roles of Rhodobacter sphaeroides copper chaperones PCuAC and Sco (PrrC) in the assembly of the copper centers of the aa3-type and the cbb3-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 aa3-type CcO containing a di-copper CuA center and mono-copper CuB, plus a cbb3-type CcO that contains CuB but lacks CuA. Three copper chaperones are located in the periplasm of R. sphaeroides, PCuAC, PrrC (Sco) and Cox11. Cox11 is required to assemble CuB of the aa3-type but not the cbb3-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in CuA assembly in mitochondria and bacteria, and with CuB assembly of the cbb3-type CcO. PCuAC is present in many bacteria, but not mitochondria. PCuAC of Thermus thermophilus metallates a CuA center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa3- and cbb3-type CcOs of R. sphaeroides has been examined in strains lacking PCuAC, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu2+. The absence of PrrC has a greater effect than the absence of PCuAC and PCuAC appears to function upstream of PrrC. Analysis of purified aa3-type CcO shows that PrrC has a greater effect on the assembly of its CuA than does PCuAC, and both chaperones have a lesser but significant effect on the assembly of its CuB even though Cox11 is present. Scenarios for the cellular roles of PCuAC and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to CuA of the aa3-type CcO and to CuB of the cbb3-type CcO, while the predominant role of PCuAC may be to capture and deliver copper to PrrC and Cox11.

Copper Starvation-inducible Protein for Cytochrome Oxidase Biogenesis in Bradyrhizobium japonicum

“…Sco1 functioned in this assay as a disulfide reductase to maintain the correct oxidation state of the subunit II cysteine ligands. In vivo data with Rhodobacter sphaeroides confirmed that PCu A C and Sco1 co-participate in the assembly of a functional Cu A center in cytochrome aa 3 (24). Intriguingly, the authors of this study noticed a role of PCu A C also in the formation of the Cu B center of cytochrome cbb 3 .…”

“…Two independent studies (Ref. 24 and this work) have now arrived at this new function. With an expedient combination of genetic and biochemical approaches, Thompson et al (24) have recently shown for R. sphaeroides that PCu A C works both for the metallation of the Cu A site of its aa 3 -type oxidase and for the metallation of the Cu B site of its cbb 3 -type oxidase.…”

“…The symbiosis defect caused by the B. japonicum scoI mutant remained enigmatic (10,21). Other reports have shown, however, that SenC and PrrC play a role in the formation of active cytochrome cbb 3 (22)(23)(24). (iv) A more recently discovered copper chaperone is PCu A C, which rivals Sco1 in its function to metallate the Cu A site.…”

Characterization of the P IB -Type ATPases Present in Thermus thermophilus

“…The genes encoding two periplasmic copper chaperones, Sco1 and PCu A C, have been identified in the genome of T. thermophilus. PCu A C appears to deliver Cu ϩ into subunit II of the T. thermophilus cytochrome ba 3 respiratory oxygen reductase (1), and a role for the equivalent protein in Rhodobacter sphaeroides in the assembly of copper-containing respiratory oxidases has also been reported (60). Similarly, the Sco1/PrrC/SenC family of proteins has been implicated in copper acquisition by bacteria and in the assembly of copper-containing proteins, including respiratory oxygen reductases (11,23,24,59,60).…”

“…PCu A C appears to deliver Cu ϩ into subunit II of the T. thermophilus cytochrome ba 3 respiratory oxygen reductase (1), and a role for the equivalent protein in Rhodobacter sphaeroides in the assembly of copper-containing respiratory oxidases has also been reported (60). Similarly, the Sco1/PrrC/SenC family of proteins has been implicated in copper acquisition by bacteria and in the assembly of copper-containing proteins, including respiratory oxygen reductases (11,23,24,59,60). Since P IB -type copper pumps have been implicated in the assembly of the copper-containing cbb 3 -type respiratory oxygen reductases (26,35), it is reasonable that the periplasmic copper chaperones present in T. thermophilus may function together with CopA and/or CopB in the assembly of the ba 3 -type and caa 3 -type copper-containing respiratory oxygen reductases present in T. thermophilus (55,61).…”