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. 2005 Jun 1;61(Pt 6):577-80.
doi: 10.1107/S1744309105014557. Epub 2005 Jun 1.

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase

Yuji Ashikawa  1 , Zui Fujimoto, Haruko Noguchi, Hiroshi Habe, Toshio Omori, Hisakazu Yamane, Hideaki Nojiri
Affiliations

Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase

Yuji Ashikawa et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 A and belongs to space group P2(1), with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 A, alpha = gamma = 90, beta = 103.8 degrees. Diffraction data for the type I crystal gave an overall Rmerge of 8.0% and a completeness of 100%. Its VM value is 2.63 A3 Da(-1), indicating a solvent content of 53.2%.

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Figures

Figure 1
Figure 1
Components and functions of the CARDO system, showing the electron-transfer reactions and conversion of carbazole to an unstable product, 1,9a-dihydroxy-1-hydrocarbazole, which has not been detected directly.
Figure 2
Figure 2
Photographs of the CARDO-OJ3 and CARDO-FCA10 complex crystals. The lines indicate 0.2 mm. (a) Type I crystals grown in 1–2 mM sodium dithionite, 14%(v/v) PEG MME 2000 and 0.05 M Bis-Tris pH 6.5. (b) Triangular plate-shaped crystals; pieces of the blossom-like crystals shown in (a). (c) Type I crystals grown in 0.1 M ammonium acetate, 12.5%(v/v) PEG 3350 and 0.05 M Bis-Tris pH 5.5. (d) Triangular plate-shaped crystals; pieces of the blossom-like crystals shown in (c).
Figure 3
Figure 3
SDS–PAGE and Western blot analysis of the dissolved crystals. (a) SDS–PAGE of the dissolved crystals (5 µg each). Lane M, low-molecular-weight markers; lane 1, dissolved crystals of CARDO-OJ3; lane 2, dissolved type I crystals of the complex; lane 3, dissolved type II crystals of the complex; lane 4, dissolved crystals of CARDO-FCA10. (b) Western blots using anti-CarAcCA10 antibody. Lane numbers are the same as in (a).
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