FIG 4 .

Bypass suppressors of R. sphaeroides mutants lacking CcoA are located in CopA and regain cbb₃-Cox activity at the expense of Cu²⁺ hypersensitivity. (A) Spontaneous NADI⁺ bypass suppressors ΔccoA Δaa₃/Rev_i that regained cbb₃-Cox activity were isolated from the ΔccoA Δaa₃ mutant. (B) The ΔccoA Δaa₃/Rev_i suppressors exhibit hypersensitivity to Cu²⁺ compared with the wild type (wt) and the ΔccoA Δaa₃ mutant. (C) The suppressor mutations corresponded to 2-bp (CG) deletions in copA (RSP_2829) of R. sphaeroides in a stretch of five CG repeats located immediately after the fourth transmembrane segment. They are compared with similar CcoA suppressors (CopA^SE8R1 and CopA^SE8R2) isolated previously from R. capsulatus (32). The latter mutations corresponded to a single-base-pair (C) indel located in a stretch of 10 C repeats of R. capsulatus CopA before its first transmembrane helix. In both species, the suppressor mutations led to translational frameshifts that abolished CopA activity, leaving intact the possibility of producing N-terminally truncated polypeptides that still carry the MBDs. The N-terminal MBD, the phosphorylation domain (DKTGT), and the transmembrane metal binding motif (CPC) are represented in CopA.