Activation of α-Keto Acid-Dependent Dioxygenases: Application of an {FeNO}⁷/{FeO₂}⁸ Methodology for Characterizing the Initial Steps of O₂ Activation

Abstract

The α-keto acid dependent dioxygenases are a major subgroup within the O2-activating mononuclear non-heme iron enzymes. For these enzymes, the resting ferrous, the substrate plus cofactor-bound ferrous, and the FeIV=O states of the reaction have been well studied. The initial O2-binding and activation steps are experimentally inaccessible and thus are not well understood. In this study, NO is used as an O2 analog to probe the effects of α-keto acid binding in 4-hydroxyphenylpyruvate dioxygenase (HPPD). A combination of EPR, UV-vis absorption, magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies in conjunction with computational models is used to explore the HPPD-NO and HPPD-HPP-NO complexes. New spectroscopic features are present in the α-keto acid bound {FeNO}7 site that reflect the strong donor interaction of the α-keto acid with the Fe. This promotes the transfer of charge from the Fe to NO. The calculations are extended to the O2 reaction coordinate where the strong donation associated with the bound α-keto acid promotes formation of a new, S=1 bridged FeIV-peroxy species. These studies provide insight into the effects of a strong donor ligand on O2 binding and activation by FeII in the α-keto acid dependent dioxygenases and are likely relevant to other subgroups of the O2 activating non-heme ferrous enzymes.

Insights into enzymatic halogenation from computational studies

“…Although building directly on their earlier hydroxylase work discussed above (Diebold et al, 2011), they did not further justify why they chose BP86 over BP86 + 10% HF in this case. Their model included some second-sphere active-site residues that are involved in hydrogen bonding to the substrate or the first-sphere complex.…”

“…They “spectroscopically calibrated” this particular method against UV/Vis absorption and EPR data of NO-bound biomimetic complexes (Schenk et al, 2004) and a hydroxylase (Diebold et al, 2011). They specifically discarded B3LYP as it yielded a qualitatively different electronic structure incompatible with the spectral data.…”

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…[29] At the same time, BP86+10%HF was selected for the investigation of the O2 reactivity of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) on the basis of calibration on NO complexes (a widely employed O2 surrogate for spectroscopic purposes), with B3LYP underestimating the charge transfer from Fe to NO. [30] Interestingly, the success of spectral predictions showed B3LYP to be appropriate for describing the electronic structure of NO bound to cysteine dioxygenase model complexes. [31] Finally, for a functional model complex of iron superoxide dismutase, modeling of absorption and Raman spectra suggested the preference for X-ray crystallographic instead of DFT-optimized geometries.…”

“…[94] On the other hand, B3LYP was supported by CCSD(T) and NEVPT2 benchmarks on small models of the proposed intermediates; [30] nevertheless, the benchmarks were carried out with B3LYP geometries, and they did not include the triplet bicyclic structure. As neither approach for validating the employed functionals refers directly to the involved O2 adducts, the question remains open until more accurate computations are available.…”

Substrate Promotes Productive Gas Binding in the α-Ketoglutarate-Dependent Oxygenase FIH

“…Nitric oxide (NO) is commonly used as an O 2 surrogate to learn about intermediates and the chemistry of non heme O 2 activating enzymes as bound NO adopts a similar geometry to that of O 2 . 16–18 NO is capable of reversibly binding to Fe 2+ within enzyme active sites, altering the electronic properties to allow characterization by conventional EPR and electronic absorption spectroscopy. 19–21 Metal-nitrosyl complexes are generally described as {MNO} n , where n represents the sum of the metal d and NO π* electrons; Fe 2+ bound NO is {FeNO} 7 22.…”