The α- and β-Subunit Boundary at the Stem of the Mushroom-Like α3β3-Type Oxygenase Component of Rieske Non-Heme Iron Oxygenases Is the Rieske-Type Ferredoxin-Binding Site

Abstract

Cumene dioxygenase (CumDO) is an initial enzyme in the cumene degradation pathway of Pseudomonas fluorescens IP01 and is a Rieske non-heme iron oxygenase (RO) that comprises two electron transfer components (reductase [CumDO-R] and Rieske-type ferredoxin [CumDO-F]) and one catalytic component (α₃β₃-type oxygenase [CumDO-O]). Catalysis is triggered by electrons that are transferred from NAD(P)H to CumDO-O by CumDO-R and CumDO-F. To investigate the binding mode between CumDO-F and CumDO-O and to identify the key CumDO-O amino acid residues for binding, we simulated docking between the CumDO-O crystal structure and predicted model of CumDO-F and identified two potential binding sites: one is at the side-wise site and the other is at the top-wise site in mushroom-like CumDO-O. Then, we performed alanine mutagenesis of 16 surface amino acid residues at two potential binding sites. The results of reduction efficiency analyses using the purified components indicated that CumDO-F bound at the side-wise site of CumDO-O, and K117 of the α-subunit and R65 of the β-subunit were critical for the interaction. Moreover, these two positively charged residues are well conserved in α₃β₃-type oxygenase components of ROs whose electron donors are Rieske-type ferredoxins. Given that these residues were not conserved if the electron donors were different types of ferredoxins or reductases, the side-wise site of the mushroom-like structure is thought to be the common binding site between Rieske-type ferredoxin and α₃β₃-type oxygenase components in ROs. IMPORTANCE We clarified the critical amino acid residues of the oxygenase component (Oxy) of Rieske non-heme iron oxygenase (RO) for binding with Rieske-type ferredoxin (Fd). Our results showed that Rieske-type Fd-binding site is commonly located at the stem (side-wise site) of the mushroom-like α₃β₃ quaternary structure in many ROs. The resultant binding site was totally different from those reported at the top-wise site of the doughnut-like α₃-type Oxy, although α₃-type Oxys correspond to the cap (α₃ subunit part) of the mushroom-like α₃β₃-type Oxys. Critical amino acid residues detected in this study were not conserved if the electron donors of Oxys were different types of Fds or reductases. Altogether, we can suggest that unique binding modes between Oxys and electron donors have evolved, depending on the nature of the electron donors, despite Oxy molecules having shared α₃β₃ quaternary structures.

Keywords: Rieske non-heme iron oxygenase; dioxygenases; electron transport; ferredoxin; protein-protein interactions.

FIG 1

Potential binding sites of CumDO-F on CumDO-O. Two potential binding sites were predicted by docking simulations using GRAMM-X. In panels A and B, CumDO-F models based on the crystal structures of the TDO-F (PDB entry 4EMJ) (37) were used, while in panel C, the CumDO-F model prepared from BDO-F (PDB entry 1FQT) (8) was used. The surfaces of the α- and β-subunits of CumDO-O (PDB entry 1WQL) are shown in magenta and green, respectively. Homology-modeled CumDO-Fs are shown in blue in the ribbon model. (A) Binding at the α-subunit interface at the top-wise site and (B and C) binding at the α- and β-subunit boundary at the side-wise site are shown.

FIG 2

Amino acid residues of CumDO-O for alanine substitution. Each of 16 amino acid residues of CumDO-O (light blue and yellow) was replaced with alanine. In both panels, the left images show the molecular surface of CumDO-O, with the positions of the substituted residues and two potential binding sites indicated by orange squares, and the right images show ribbon models of potential binding sites, in which alanine-substituted residues are shown in sticks. Lys33, Leu35, Arg39, and Arg407 of α subunit 1 (α₁ subunit) and Asp158, Trp159, Leu162, and Glu180 of neighboring α subunit 2 (α₂ subunit) were located at the top-wise site (A). Lys117 and Lys141 of the α₁ subunit, Leu241, Asp253, and Lys258 of the α₂ subunit, Arg65 of β subunit 1 (β₁ subunit), and Leu98 and Trp100 of the neighboring β subunit 2 (β₂ subunit) were located at the side-wise site (B). α- and β-subunits of CumDO-O are shown in magenta and green, respectively.

FIG 3

Reduction efficiencies of alanine-substituted CumDO-Os by CumDO-F. Panels A and B show the reduction efficiencies of single-alanine-substituted CumDO-Os and double-alanine-substituted CumDO-Os, respectively. Results for WT CumDO-O (set at 100%) are shown as gray bars. Black and white bars are the reduction efficiencies of CumDO-O derivatives with amino acid substitutions at the top-wise and side-wise potential CumDO-F-binding sites, respectively. Error bars indicate standard deviations from three independent experiments. The data were assessed using Student's t test with P values of <0.005 (**) or <0.001 (***) for alanine-substituted CumDO-Os compared to the WT.

FIG 4

Conserved charged residues in the structures of α₃β₃-type Oxy components. Structures of different α₃β₃-type Oxys of 10 RO systems are shown: (A) CumDO from P. fluorescens IP01 (PDB entry 1WQL), (B) TDO from P. putida F1 (PDB entry 3EN1), (C) BDO from R. jostii RHA1 (PDB entry 1ULI), (D) BDO from P. pnomenusa B-356 (PDB entry 3GZY), (E) BDO from B. xenovorans LB400 (PDB entry 2XR8), (F) BDO from Sphingomonas yanoikuyae B1 (PDB entry 2GBX), (G) NDO from Pseudomonas sp. strain NCIB9816-4 (PDB entry 1NDO), (H) NDO from Pseudomonas sp. strain C18 (PDB entry 4HJL), (I) NBDO from Comamonas sp. strain JS765 (PDB entry 2BMO), and (J) PAH-hydroxylating dioxygenase from Sphingomonas sp. strain CHY-1 (PDB entry 2CKF) are shown in the side view. α- and β-subunits are shown in magenta and green, respectively. The groove of the potential CumDO-F binding site is shown in orange in panel A. Conserved positive-charged residues are in yellow.

FIG 5

Amino acid sequence alignments of α- and β-subunits of 10 structure-solved Oxys. Parts of amino acid sequence alignments of Oxy components of 10 RO systems are shown. Enzyme names followed by origins (subscripted) are shown at left. Alanine-substituted residues (arrowheads) at the top-wise and side-wise sides are shown by light blue and yellow backgrounds, respectively. Conserved positive amino acid residues in the side-wise putative Fd-binding site (Fig. 4) are shown in light blue and light green in the α- and β-subunit sequences, respectively. Cys and His residues in the α-subunit sequences involved in the coordination of Rieske clusters are shown in red. Numbers at termini show the positions of terminal amino acid residues in CumDO-O protein.

FIG 6

Amino acid sequence alignments of α- and β-subunits of CumDO Oxy with those of α₃β₃-type Oxys coupled with Rieske-type Fds. Only the parts of Oxy including the potential important amino acid residues for binding to Rieske-type Fds are shown. Conserved and similar amino acid residues at positions crucial for binding between α₃β₃-type Oxys and Rieske-type Fds (indicated by arrowheads) are darkly and lightly shaded, respectively. Four ligands for the Rieske cluster in α subunits are underlined. Numbers at right show the positions of terminal amino acid residues. Enzyme names followed by origins (subscripted) are shown at left.

FIG 7

Surface electrostatic potential of seven structure-solved Rieske-type Fds. Surface electrostatic potential of (A) BDO-F from B. xenovorans LB400 (PDB entry 1FQT), (B) BDO-F from B. xenovorans B1 (PDB entry 2I7F), (C) BDO-F from Acidovorax sp. strain KKS102 (PDB entry 2E4P), (D) TDO-F from P. putida F1 (PDB entry 3DQY), (E) NDO-F from Pseudomonas sp. strain NCIB9816-4 (PDB entry 2QPZ), (F) CARDO-F from P. resinovorans CA10 (PDB entry 1VCK), and (G) CARDO-F from Nocardioides aromaticivorans IC177 (PDB entry 3GCE) are shown. Fds in panels A to E transfer electrons to α₃β₃-type Oxys, and Fds in panels F and G transfer electrons to α₃-type Oxys. Fd molecules in the middle are facing the binding surfaces of Oxy molecules, and Rieske clusters are located at their tips. Positive and negative potential regions are shown in blue and red, respectively. Positions of conserved Asp/Glu residues are also shown.

FIG 8

Amino acid sequence alignment of structure-solved Rieske-type Fds. The positions of two negatively charged Glu residues (Glu55 and Glu64), reported to mediate electrostatic interactions with positively charged amino acid residues of Oxy in the Oxy-Fd complex crystal of CARDO (18) are shown by black arrowheads and the corresponding Asp/Glu residues in other Fds by gray arrowheads. Amino acid residues identical or similar to CumDO-F (CumA3 protein) in other Fds are shaded in black and gray, respectively. The four conserved ligands for the Rieske cluster are underlined. Numbers at right show the positions of terminal amino acid residues. Protein names of Fds and their origins (subscripted) are shown at left.

FIG 9

Possible electron transfer pathway from the Rieske cluster of CumDO-F to the non-heme iron of CumDO-O. The hydrogen bonds that mediate the electron transfer are indicated by red dashed lines. The atoms involved in the electron transfer and in the coordination with the iron ions are shown in a stick model. Nitrogen, oxygen, sulfur, and iron atoms are colored blue, red, yellow, and orange, respectively. Carbon atoms in CumDO-F are colored cyan, and those in α-subunits 1 and 2 of CumDO-O are colored green and brown, respectively. Distances among three electron transfer centers, two Rieske clusters, and an active site iron are shown by black dotted lines.