Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1.
The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE). The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3). Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3). If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.
Niebisch
Axel
A
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
Bott
Michael
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003352:Corynebacterium / Q000201:enzymology*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D014450:Electron Transport Complex III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D006418:Heme; D010088:Oxidoreductases; C020094:duroquinol oxidase; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III