The Apparently Unreactive Substrate Facilitates the Electron Transfer for Dioxygen Activation in Rieske Dioxygenases

Rieske dioxygenases belong to the non‐heme iron family of oxygenases and catalyze important cis‐dihydroxylation as well as O‐/N‐dealkylation and oxidative cyclization reactions for a wide range of substrates. The lack of substrate coordination at the non‐heme ferrous iron center, however, makes it particularly challenging to delineate the role of the substrate for productive [Formula: see text] activation. Here, we studied the role of the substrate in the key elementary reaction leading to [Formula: see text] activation from a theoretical perspective by systematically considering (i) the 6‐coordinate to 5‐coordinate conversion of the non‐heme Fe(II) upon abstraction of a water ligand, (ii) binding of [Formula: see text] , and (iii) transfer of an electron from the Rieske cluster. We systematically evaluated the spin‐state‐dependent reaction energies and structural effects at the active site for all combinations of the three elementary processes in the presence and absence of substrate using naphthalene dioxygenase as a prototypical Rieske dioxygenase. We find that reaction energies for the generation of a coordination vacancy at the non‐heme Fe [Formula: see text] center through thermoneutral H(2)O reorientation and exothermic [Formula: see text] binding prior to Rieske cluster oxidation are largely insensitive to the presence of naphthalene and do not lead to formation of any of the known reactive Fe‐oxygen species. By contrast, the role of the substrate becomes evident after Rieske cluster oxidation and exclusively for the 6‐coordinate non‐heme Fe [Formula: see text] sites in that the additional electron is found at the substrate instead of at the iron and oxygen atoms. Our results imply an allosteric control of the substrate on Rieske dioxygenase reactivity to happen prior to changes at the non‐heme Fe [Formula: see text] in agreement with a strategy that avoids unproductive [Formula: see text] activation.