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

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin Fe^III–(hydro)­peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native Fe^II/O₂ reaction is evaluated. For the native Fe^II/O₂ reaction, an Fe^III–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of Fe^III–superoxo and Fe^III–hydroperoxo species in nonheme Fe biochemistry

Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class Ia Binuclear Non-Heme Iron Enzyme

The class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i> (<i>Ct</i>) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O₂. This reaction produces a stable Mn^IVFe^III cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn^IVFe^III cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe^III, whereas MCD reflects the spin-allowed transitions mostly on the Mn^IV. We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn^IV at the site proximal to Phe₁₂₇. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d–d and oxo and OH^– to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn^IVFe^III cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the Mn^IV. From DFT calculations, the Mn^IV at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH^– terminal ligand on this Mn^IV provides a high proton affinity that could gate radical translocation to the α (R1) subunit