Thermodynamic properties of metal centers in mononuclear non-heme iron enzymes determine their ability to activate C-H bonds. These structurally diverse enzymes are involved in a broad spectrum of biological functions, but are not as well-characterized as their heme analogs. α-ketoglutarate (αKG) dependent hydroxylases, such as αKG-dependent taurine hydroxylase (TauD), represent the best-studied class of non-heme Fe enzymes and are believed to utilize a P450-like hydroxyl rebound mechanism, whereby initial hydrogen atom transfer from a substrate to the non-heme Fe(IV)=O is followed by transfer of the resulting ●OH group to the substrate. As in its heme analogues, C-H bond activation by TauD is carried out with high specificity, avoiding oxidative self-damage. This ability is determined by the Gibbs free energy of the Fe-OH bond formation and is contributed by the redox potential of the oxidizing species and the proton affinity of the reduced species, as described by the Bordwell thermodynamic cycle. Details of such a balance between the E°' of the Fe(IV)=O/Fe(III)-O− couple in non-heme iron centers and the pKa of the corresponding Fe(III)-OH/Fe(III)-O− species are not yet understood. Synthetic modeling studies have suggested that the non-heme Fe(III)-O(H) species is highly basic with a pKa of ~25. However, pKa’s of analogous species in heme proteins are much lower (&lt;12). A time-resolved Raman study on TauD suggests that the pKa of its Compound II-like species must be less than 8, resulting in the population of Fe(III)-O instead of Fe(III)-OH as expected for the hydroxyl rebound mechanism. Here we report the development of in situ models of the ferric TauD species in complexes with natural co-substrates as the in situ mimic of the transient Fe(III)-O(H) F3 species. We report on the redox properties of Fe(II/III) TauD in several coordination states using redox difference FTIR spectro-electrochemistry. We characterize vibrational changes attributable to the Fe(II/III)-TauD redox-dependent conformations at the iron binding site with and without co-substrates (αKG and taurine). Normal pulse voltammetric titrations are used to directly determine the Fe(II/III) E½. We assess protonation states and the likely pKa of the Fe(III)-OH species from pH sensitivity and available structural data. A striking hysteresis between the reduction and oxidation steps strongly implies involvement of an additional redox-linked structural change at TauD’s active site.

Protonation states of oxygenic ligands in the non-heme iron dioxygenase, TauD

251st National Meeting (2016)

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