Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB

Inaba et al. 10.1073/pnas.0507570103.

Supporting Information

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7

Supporting Figure 5

Fig. 5. Two reaction pathways in DsbB-catalyzed DsbA oxidation. The rapid pathway proceeds through direct disulfide exchanges between the active site cysteines, in which UQ drives the reaction by oxidizing Cys-41 and Cys-44. The slow pathway proceeds through the disulfide-linked DsbA-DsbB complex, which resolves slowly, in a UQ-dependent manner. The end results in the quinone-coupled reactions are the regeneration of both DsbA and DsbB as their oxidized and active forms. The commitment steps for the two pathways are supposed to be in the nucleophilic attacks (shown by red broken arrows) by Cys-33 of DsbA (rapid pathway) and by Cys-130 of DsbB (slow pathway) of respective target cysteines (DsbA Cys-30 and DsbB Cys-41, respectively). Note that DsbB-bound quinones in the slow pathway assume the electronic transition states that are induced by reduced Cys-44 (ref. 1; shown in pink). The rapid pathway may also involve a brief quinone transition (2). This figure was originally presented in ref. 3.

1. Inaba, K., Takahashi, Y. H., Fujieda, N., Kano, K., Miyoshi, H. & Ito, K. (2004) J. Biol. Chem. 279, 6761-6768.

2. Takahashi, Y.-h., Inaba, K. & Ito, K. (2004) J. Biol. Chem. 279, 47057-47065.

3. Inaba, K., Takahashi, Y.-h. & Ito, K. (2005) J. Biol. Chem. 280, 33035-33044.

Supporting Figure 6

Fig. 6. Quantification of DsbB-bound UQ. DsbB preparations having the indicated amino acid substitution were extracted with organic solvent as described previously (1), followed by Shim-pack VP-ODS reverse phase column chromatography (Shimadzu), in which quinone species were eluted with methanol-isopropanol (3:1) at a flow rate of 0.2 ml/min. The UQ peaks at elution time of 12.8 min, verified by electrospray ionization-MS, were quantified from the HPLC chromatograms (2). The numbers nearby the elution peaks indicate relative UQ contents.

1. Takahashi, Y.-h., Inaba, K. & Ito, K. (2004) J. Biol. Chem. 279, 47057-47065.

2. Inaba, K., Takahashi, Y.-h. & Ito, K. (2005) J. Biol. Chem. 280, 33035-33044.

Supporting Figure 7

Fig. 7. HPLC elution profiles of organic solvent extracts of various DsbB preparations. Samples obtained from DsbB preparations having the indicated amino acid substitution(s) were separated as described in Fig. 6. The numbers nearby the elution peaks indicate relative UQ contents.