Fig. 4.

All 4 cysteine motifs are required for CcoG function. (A) A cartoon of CcoG showing its Cu and [4Fe-4S] cluster-binding motifs as red boxes. The single mutants were derivatives of pRKccoG_FLAG in R. capsulatus (SI Appendix, Table S2) and the Cys-to-Ala mutations (C₁₁₀A, C₂₂₁A, C₂₆₇A, and C₂₉₁A) are indicated by arrows. The quadruple mutants were derivatives of pBADccoG in E. coli (SI Appendix, Table S2); the CcoG C₁₁₀, C₁₁₄, C₂₁₇, and Cys₂₂₁ residues replaced with Ser are indicated by green stars in the case of CcoG(ΔCu), and the C₂₆₇, C₂₇₀, C₂₉₁, and C₂₉₄ residues replaced with Ser indicated by purple stars in the case of CcoG(ΔFeS) are shown. (B) The strains described in A were tested for growth on enriched MPYE plates after incubation at 35 °C for about 20 h. A representative set is shown (n = 3). (C) The cbb₃-Cox activity of the indicated strains was determined as in Fig. 3A. Each activity was measured at least twice in at least 3 independent experiments (n = 3). The cbb₃-Cox activity exhibited by the wild-type strain was set to 100%. Statistical analysis was performed on the raw data using the Student’s t test with P < 0.0003 as the level of significance between the wild type and all strains, with the exception of the ΔccoG-pRKccoGC267A (P < 0.0007) and ΔccoG-pRKccoG (P > 0.05). Data are presented as means ± SD. (D) Immunoblot showing that the Cys-to-Ala substitution mutant derivatives of CcoG are produced in R. capsulatus (50 µg of membranes was loaded).