Electron transfer kinetics during the reduction and turnover of the cytochrome caa3 complex from Bacillus subtilis.
The cytochrome caa3 complex from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory enzyme complexes. The key difference in the cytochrome caa3 complex lies in the addition of a domain, homologous with mitochondrial cytochrome c, that is fused to the C-terminal end of its subunit II. Measurements of steady-state and transient reduction kinetics have been carried out on the cytochrome caa3 complex. Reduction of the cyanide-bound enzyme with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) supports a sequence of electron transfer in which cytochromec is reduced initially, and this is followed by rapid internal electron transfer from cytochrome c to CuA and from CuA to cytochrome a. Steady-state kinetics with exogenous cytochrome c as the substrate demonstrates the capability of the cytochrome caa3 complex to act as a cytochrome c oxidase. The cytochrome c from B. subtilis is the most efficient cytochrome c of those tested. Steady-state kinetics with ascorbate-TMPD as the reductant, in the absence of exogenous cytochrome c, reveals a biphasic pattern even though only a single, covalent cytochrome c interaction site is present. The two-phase kinetics are characterized by a low activity phase associated with a high apparent affinity for TMPD and a high activity phase with a low affinity for TMPD. This pattern is observed over a wide range of ionic strengths and enzyme concentrations, and with both purified and membrane extract forms of cytochrome caa3. It is proposed that the biphasic steady-state kinetics of this oxidase, and other members of the cytochrome oxidase superfamily, do not result directly from different interactions with cytochrome c but are due to a change in the redox kinetics within the centers of the conventional oxidase unit itself. Our results will be related to models that account for the biphasic steady-state kinetics exhibited by cytochrome oxidase.
Assempour
M
M
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Lim
D
D
Hill
B C
BC
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D003486:Cyanides / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008956:Models, Chemical; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D003486:Cyanides; D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine