The Putative Assembly Factor CcoH Is Stably Associated with the cbb ₃ -Type Cytochrome Oxidase

Abstract

Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb 3 -type cytochrome c oxidases (cbb 3 -Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb 3 -Cox stability. Biogenesis of cbb 3 -Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb 3 -Cox. Most bacteria expressing cbb 3 -Cox also contain the ccoGHIS genes, which encode putative cbb 3 -Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb 3 -Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (N out -C in ) topology. In its absence, neither the fully assembled cbb 3 -Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb 3 -Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb 3 -Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb 3 -Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb 3 -Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb 3 -Cox than an assembly factor per se.

Bacterial Adaptation of Respiration from Oxic to Microoxic and Anoxic Conditions: Redox Control

“…Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, generates the active 230 kDa complex (126). Recent results have proposed that CcoH behaves more similar to a subunit of the cbb 3 oxidase rather than a transient assembly factor per se (173). The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb 3 -type oxidase (126).…”

A Copper Relay System Involving Two Periplasmic Chaperones Drives cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus

“…45 In addition, the assembly factor CcoH is stably associated with the cbb 3 -Cox complex in Rhodobacter membranes. 46 …”

Missense Mutations in Cytochrome c Maturation Genes Provide New Insights into Rhodobacter capsulatus cbb 3 -Type Cytochrome c Oxidase Biogenesis

“…We note that these two processes are distinct and, conceivably, that different steady-state amounts of CcoN might be found in the membranes, depending on the nature of the mutation(s). Because CcoN together with CcoO forms a stable CcoNO subcomplex (8,32), it appears that CcoN awaits the completion of its own maturation process when CcoO is absent. Conversely, when a mature CcoO protein is produced, its association with immature CcoN (as occurs in the absence of CcoA [11] or CcoI [23]) presumably leads to its degradation.…”