Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides.
The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc(1) complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc(1) complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 degrees C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Angstroms resolution.
Elberry
Maria
M
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, 74078, USA.
Xiao
Kunhung
K
Esser
Lothar
L
Xia
Di
D
Yu
Linda
L
Yu
Chang-An
CA
D002152:Calorimetry, Differential Scanning; D003460:Crystallization; D018360:Crystallography, X-Ray; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015202:Protein Engineering; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics
D014450:Electron Transport Complex III