Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites

Abstract

FeIII-(hydro)peroxy intermediates have been isolated in two classes of mononuclear non-heme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here, we present the NRVS spectra of side-on FeIII-peroxy and end-on FeIII-hydroperoxy model complexes and assign these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe-O-O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of FeIII-peroxy intermediates in mononuclear non-heme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity.

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native FeII O2 Reaction

“…Due to its isotope sensitivity, the 510 cm −1 feature can be assigned as the νFe−O. Qualitatively, based on a previous NRVS study on high-spin Fe III − (hydro)peroxy models, 50 the peaks centered at 277 cm −1 are equatorial Fe−N His stretches. This previous study showed that the energy of the νFe−O and the splitting between equatorial Fe−N stretches are sensitive to the coordination mode of O 2 and its protonation state.…”

“…49 To calibrate our DFT method for BZDOp and define the binding mode and protonation state of high-spin Fe III –(hydro)peroxy models, we previously analyzed NRVS data on a side-on Fe III –peroxy model 10 and an end-on Fe III –hydroperoxy model 10 (both high-spin and ligated by the 4-coordinate 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane (TMC) ligand). 50 …”

“…49 To calibrate our DFT method for BZDOp and define the binding mode and protonation state of high-spin Fe III − (hydro)peroxy models, we previously analyzed NRVS data on a side-on Fe III −peroxy model 10 . 50 In the present study, we first present the NRVS data of BZDOp and assign its structure based on DFT calculations calibrated to the high-spin Fe III (hydro)peroxy models. We then define its reactivity with substrate, including consideration of whether BZDOp can be in an equilibrium with an Fe III − superoxo reduced Rieske species.…”

“…This methodology has previously been applied to the low-spin Fe III hydroperoxy intermediate in bleomycin (BLM), the Fe IV‑ oxo intermediate in SyrB2 and the bridged peroxide intermediate P′ in the binuclear Fe site of AurF . To calibrate our DFT method for BZDOp and define the binding mode and protonation state of high-spin Fe III –(hydro)­peroxy models, we previously analyzed NRVS data on a side-on Fe III –peroxy model and an end-on Fe III –hydroperoxy model (both high-spin and ligated by the 4-coordinate 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane (TMC) ligand) …”

NRVS for Fe in Biology: Experiment and Basic Interpretation

“…Besides non-Rieske [2Fe2S] 22 and [3Fe4S] 23 proteins and [4Fe4S] 24 proteins, It is important to note that 57 Fe NRVS has been expanded to study electron transfer in larger enzymes and their catalytic metallocofactors such as (but not comprehensively) the NiFe site in [NiFe] hydrogenase, 3, 25–26 the diiron site in [FeFe] hydrogenase, 27–28 the P [8Fe7S]- and M [7Fe9SMo]- clusters of Mo-Nitrogenase, 29–32 heme systems, 33–34 non-heme systems, 35–36 and nitric-oxide sensing [4Fe4S] clusters. 37 Further as the systems of interest have become more complicated, with multiple FeS clusters of differing composition, innovative strategies of site-specific 57 Fe labeling have been implemented that target maturation 28 or a modified cofactor insertion.…”