The α- and β-Subunit Boundary at the Stem of the Mushroom-Like α ₃ β ₃ -Type Oxygenase Component of Rieske Non-Heme Iron Oxygenases Is the Rieske-Type Ferredoxin-Binding Site

Abstract

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 α 3 β 3 quaternary structure in many ROs.

Rieske Oxygenases and Other Ferredoxin‐Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

“…Sequence alignment with other ROs indicate that these two positive charged residues presumably play an essential role in the side-wise protein interaction between RO-Fds and heterohexameric RO-Oxys (α 3 β 3 ). [135] Unlike described charge-transfer complexes in three-component ROs, considerably less structural information on the interaction between RO-Reds and their associated RO-Oxys is reported. To the best of our knowledge, the only example in the literature described is the complex between NdmA and the Fd-domain of the associated RO-Red NdmD (NdmD Fd , PDB: 6ICM).…”

“…[24,134] Recently, specific residues at the surfaces of the heterohexameric RO-Oxy (α 3 β 3 ) and associated RO-Fd of CDO from P. fluorescens IP01 have been determined as crucial for electron transfer between the corresponding Rieske [2Fe-2S] clusters. [135] Thereby, docking studies in combination with alanine scanning revealed two residues crucial for RO-Oxy reduction. As reported, no reduction of the [2Fe-2S] cluster within the Rieske domain of RO-Oxy was observed when exchanging Lys117 in the αsubunit and Arg65 in the β-subunit.…”

Engineering Rieske oxygenase activity one piece at a time

“…The β subunits of the α 3 β 3 enzymes sit more than 10 Å away from the active site. Residues implicated in binding the reductase are shown in an oval and highlighted in purple [ 49 ]. (b) The α 3 α 3 architecture of PDO reveals that the trimers are staggered and likely stabilized by additional secondary structures found in each α subunit (dark blue and orange).…”

“…Other reports suggest that the β subunits could be involved in dictating the substrate scopes and preferences of different Rieske oxygenases [ 46 – 48 ]. Most recently, residues from both the α and β subunits were reported to mediate interactions with the ferredoxin component of a dedicated partner reductase system [ 49 ]. Mapping of these residues onto naphthalene dioxygenase (NDO) reveals that the reductase, as observed in the α 3 enzymes, also sits at the subunit–subunit interfaces ( Figure 2a , d ).…”

“…Through these experiments, several trends regarding the importance of thus far underappreciated protein regions of interest have emerged. The additional α and β subunits, as described for thermophilic proteins, likely provide an increased number of weak interactions to stabilize protein structure or create a better interaction with the needed electron donor to facilitate catalysis [ 13 , 32 , 44 , 49 ]. The entrance tunnels of Rieske oxygenases interact with substrate, geometrically position substrate for catalysis, and are designed to gate access of non-substrate molecules into the active site [ 4 , 15 , 37 ].…”