Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase.
The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.
Hakvoort
T B
TB
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
Moolenaar
K
K
Lankvelt
A H
AH
Sinjorgo
K M
KM
Dekker
H L
HL
Muijsers
A O
AO
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003576:Electron Transport Complex IV / Q000302:isolation & purification* / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*
D003576:Electron Transport Complex IV; D001205:Ascorbic Acid