Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.
A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.
Reincke
B
B
Institute of Biophysical Chemistry, Biozentrum, J.W. Goethe-Universität, D-60439, Frankfurt, Germany.
Thöny-Meyer
L
L
Dannehl
C
C
Odenwald
A
A
Aidim
M
M
Witt
H
H
Rüterjans
H
H
Ludwig
B
B
D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids
D003574:Cytochrome c Group; C083951:cytochrome c553; C022302:cytochrome C-552; D003576:Electron Transport Complex IV