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

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 (alpha(3)beta(3)-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 alpha-subunit and R65 of the beta-subunit were critical for the interaction. Moreover, these two positively charged residues are well conserved in alpha(3)beta(3)-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 alpha(3)beta(3)-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 alpha(3)beta(3) quaternary structure in many ROs. The resultant binding site was totally different from those reported at the top-wise site of the doughnut-like alpha(3)-type Oxy, although alpha(3)-type Oxys correspond to the cap (alpha(3) subunit part) of the mushroom-like alpha(3)beta(3)-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 alpha(3)beta(3) quaternary structures. 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 alpha(3)beta(3) quaternary structure in many ROs.