The putative assembly factor CcoH is stably associated with the cbb3-type cytochrome oxidase.
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.
Institut für Biochemie und Molekularbiologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
Grzegorz
G
Pawlik
Carmen
C
Kulajta
Ilie
I
Sachelaru
Sebastian
S
Schröder
Barbara
B
Waidner
Petra
P
Hellwig
Fevzi
F
Daldal
Hans-Georg
HG
Koch
D001426:Bacterial Proteins / Q000378:metabolism; D019069:Cell Respiration / Q000502:physiology; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D010957:Plasmids; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D021122:Protein Subunits; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV