Missense Mutations in Cytochrome c Maturation Genes Provide New Insights into Rhodobacter capsulatus cbb3-Type Cytochrome c Oxidase Biogenesis

摘要

The Rhodobacter capsulatus cbb3-type cytochrome c oxidase (cbb3-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb3-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb3-Cox unless the growth medium is supplemented with copper. In this work, we isolated “Cu-unresponsive” derivatives of a ccoA deletion strain that exhibited no cbb3-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb3-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c1 and c2 than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb3-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme.