Structural and Spectroscopic Properties of the Peroxodiferric Intermediate of <i>Ricinus communis</i> Soluble Δ⁹ Desaturase

Large-scale quantum and molecular mechanical methods (QM/MM) and QM calculations were carried out on the soluble Δ⁹ desaturase (Δ⁹D) to investigate various structural models of the spectroscopically defined peroxodiferric <b>(P</b>) intermediate. This allowed us to formulate a consistent mechanistic picture for the initial stages of the reaction mechanism of Δ⁹D, an important diferrous nonheme iron enzyme that cleaves the C–H bonds in alkane chains resulting in the highly specific insertion of double bonds. The methods (density functional theory (DFT), time-dependent DFT (TD-DFT), QM­(DFT)/MM, and TD-DFT with electrostatic embedding) were benchmarked by demonstrating that the known spectroscopic effects and structural perturbation caused by substrate binding to diferrous Δ⁹D can be qualitatively reproduced. We show that structural models whose spectroscopic (absorption, circular dichroism (CD), vibrational and Mössbauer) characteristics correlate best with experimental data for the <b>P</b> intermediate correspond to the μ-1,2-O₂^2– binding mode. Coordination of Glu196 to one of the iron centers (Fe_B) is demonstrated to be flexible, with the monodentate binding providing better agreement with spectroscopic data, and the bidentate structure being slightly favored energetically (1–10 kJ mol^–1). Further possible structures, containing an additional proton or water molecule are also evaluated in connection with the possible activation of the <b>P</b> intermediate. Specifically, we suggest that protonation of the peroxide moiety, possibly preceded by water binding in the Fe_A coordination sphere, could be responsible for the conversion of the <b>P</b> intermediate in Δ⁹D into a form capable of hydrogen abstraction. Finally, results are compared with recent findings on the related ribonucleotide reductase and toluene/methane monooxygenase enzymes

Structural and Spectroscopic Properties of the Peroxodiferric Intermediate of <i>Ricinus communis</i> Soluble Δ⁹ Desaturase

Large-scale quantum and molecular mechanical methods (QM/MM) and QM calculations were carried out on the soluble Δ⁹ desaturase (Δ⁹D) to investigate various structural models of the spectroscopically defined peroxodiferric <b>(P</b>) intermediate. This allowed us to formulate a consistent mechanistic picture for the initial stages of the reaction mechanism of Δ⁹D, an important diferrous nonheme iron enzyme that cleaves the C–H bonds in alkane chains resulting in the highly specific insertion of double bonds. The methods (density functional theory (DFT), time-dependent DFT (TD-DFT), QM­(DFT)/MM, and TD-DFT with electrostatic embedding) were benchmarked by demonstrating that the known spectroscopic effects and structural perturbation caused by substrate binding to diferrous Δ⁹D can be qualitatively reproduced. We show that structural models whose spectroscopic (absorption, circular dichroism (CD), vibrational and Mössbauer) characteristics correlate best with experimental data for the <b>P</b> intermediate correspond to the μ-1,2-O₂^2– binding mode. Coordination of Glu196 to one of the iron centers (Fe_B) is demonstrated to be flexible, with the monodentate binding providing better agreement with spectroscopic data, and the bidentate structure being slightly favored energetically (1–10 kJ mol^–1). Further possible structures, containing an additional proton or water molecule are also evaluated in connection with the possible activation of the <b>P</b> intermediate. Specifically, we suggest that protonation of the peroxide moiety, possibly preceded by water binding in the Fe_A coordination sphere, could be responsible for the conversion of the <b>P</b> intermediate in Δ⁹D into a form capable of hydrogen abstraction. Finally, results are compared with recent findings on the related ribonucleotide reductase and toluene/methane monooxygenase enzymes

CD/MCD/VTVH-MCD Studies of <i>Escherichia coli</i> Bacterioferritin Support a Binuclear Iron Cofactor Site

Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe­(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe­(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in <i>Escherichia coli</i> Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O₂ oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O₂ cycling supports a mechanism in which the binuclear site acts as a cofactor for the O₂ reaction, while the mononuclear site binds the substrate Fe­(II) that, after its oxidation to Fe­(III), migrates to the mineral core

Characterization of Metastable Intermediates Formed in the Reaction between a Mn(II) Complex and Dioxygen, Including a Crystallographic Structure of a Binuclear Mn(III)–Peroxo Species

Transition-metal peroxos have been implicated as key intermediates in a variety of critical biological processes involving O₂. Because of their highly reactive nature, very few metal–peroxos have been characterized. The dioxygen chemistry of manganese remains largely unexplored despite the proposed involvement of a Mn–peroxo, either as a precursor to, or derived from, O₂, in both photosynthetic H₂O oxidation and DNA biosynthesis. These are arguably two of the most fundamental processes of life. Neither of these biological intermediates has been observed. Herein we describe the dioxygen chemistry of coordinatively unsaturated [Mn^II(S^Me2N₄(6-Me-DPEN))] ⁺ (<b>1</b>), and the characterization of intermediates formed en route to a binuclear mono-oxo-bridged Mn­(III) product {[Mn^III(S^Me2N₄(6-Me-DPEN)]₂(μ-O)}²⁺ (<b>2</b>), the oxo atom of which is derived from ¹⁸O₂. At low-temperatures, a dioxygen intermediate, [Mn­(S^Me2N₄(6-Me-DPEN))­(O₂)]⁺ (<b>4</b>), is observed (by stopped-flow) to rapidly and irreversibly form in this reaction (<i>k</i>₁(−10 °C) = 3780 ± 180 M^–1 s^–1, Δ<i>H</i>₁^⧧ = 26.4 ± 1.7 kJ mol^–1, Δ<i>S</i>₁^⧧ = −75.6 ± 6.8 J mol^–1 K^–1) and then convert more slowly (<i>k</i>₂(−10 °C) = 417 ± 3.2 M^–1 s^–1, Δ<i>H</i>₂^⧧ = 47.1 ± 1.4 kJ mol^–1, Δ<i>S</i>₂^⧧ = −15.0 ± 5.7 J mol^–1 K^–1) to a species <b>3</b> with isotopically sensitive stretches at ν_O–O(Δ¹⁸O) = 819(47) cm^–1, <i>k</i>_O–O = 3.02 mdyn/Å, and ν_Mn–O(Δ¹⁸O) = 611(25) cm^–1 consistent with a peroxo. Intermediate <b>3</b> releases approximately 0.5 equiv of H₂O₂ per Mn ion upon protonation, and the rate of conversion of <b>4</b> to <b>3</b> is dependent on [Mn­(II)] concentration, consistent with a binuclear Mn­(O₂^2–) Mn peroxo. This was verified by X-ray crystallography, where the peroxo of {[Mn^III(S^Me2N₄(6-Me-DPEN)]₂(<i>trans</i>-μ-1,2-O₂)}²⁺ (<b>3</b>) is shown to be bridging between two Mn­(III) ions in an <i>end-on trans</i>-μ-1,2-fashion. This represents the <i>first characterized example of a binuclear Mn­(III)–peroxo</i>, and a rare case in which more than one intermediate is observed en route to a binuclear μ-oxo-bridged product derived from O₂. Vibrational and metrical parameters for binuclear Mn–peroxo <b>3</b> are compared with those of related binuclear Fe– and Cu–peroxo compounds

Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O₂ Activation

Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O₂ to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O₂ or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O₂ activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O₂ reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O₂ by the binuclear non-heme iron enzymes

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

Fe^III-(hydro)­peroxy intermediates have been isolated in two classes of mononuclear nonheme 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 Fe^III-peroxy and end-on Fe^III-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 Fe^III-peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity

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