Electron Transfer Complex Formation between Oxygenase and Ferredoxin Components in Rieske Nonheme Iron Oxygenase System

Abstract

Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 A resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…31) However, we succeeded in determining the carbazole-binding and carbazole/ oxygen-binding structures under oxidized and reduced conditions using a CARDO-F CA10 :CARDO-O J3 complex crystal 37) (see section IV below). Upon carbazole binding, amino acid residues Leu202-Thr214 and Asp229-Val238, which were situated near the entrance of the substrate-binding pocket, adopted different structural conformations (Fig.…”

“…We have to date succeeded in determining the crystal structures of eight components, not including CARDO-R IC177 (Fig. 6 37) succeeded in determining the crystal structure of the electron-transfer complex between CARDO-O J3 and CARDO-F CA10 (Fig. 7A).…”

“…To my knowledge, the report of the CARDO-F CA10 :CARDO-O J3 binary complex structure 37) was the first report of the position of the binding site in the molecular surface of RO components. Considering the similarity of the three classes of CARDO-O molecules, it is reasonable to assume that CARDO-F can bind to the depression in class IIB CARDO-O, which enables the Rieske cluster in CARDO-F IC177 to transfer electrons to that in CARDO-O IC177 .…”

“…CARDO-R 37) and Electrostatic and Hydrophobic Interactions Observed in a Binary Complex of CARDO-F and CARDO-O in a Class III System (B) 37,43) and Those Proposed for a Class IIB System (C). 43) In panel A, the three CARDO-O J3 subunits (chain A, red; chain B, forest green; and chain C, blue) and the three CARDO-F CA10 molecules (chain D, orange; chain E, cyan; and chain F, magenta) are shown.…”

“…In panel B, the amino acid residues involved in binding are mapped on the molecular surfaces of CARDO-F CA10 and CARDO-O J3 , based on the CARDO-F CA10 :CARDO-O J3 binary complex structure (PDB entry, 2DE5). 37) In panel C, the residues predicted to be involved in the interactions between CARDO-F IC177 and CARDO-O IC177 , based on the results of amino acid sequence alignments with CARDO-F CA10 and CARDO-O J3 , are shown.…”