Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes

Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm−1 to +32 cm−1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-μ-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.

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“…19 The software diagonalizes the electronic terms of the spin Hamiltonian:

H = J S_{1} \cdot S_{2} + H_{1} + H_{2} + H_{dip} H_{i} = bold-italicβ S_{i} \cdot g_{i} \cdot boldB + S_{i} \cdot D_{i} \cdot S_{i} + S_{i} \cdot A_{i} \cdot I_{i} - g_{n} β_{n} boldB \cdot I_{i} + I_{i} \cdot P_{i} \cdot I_{i} false(i = 1, 2 false) {boldS}_{i} \cdot {boldD}_{i} \cdot {boldS}_{i} = D_{i} [S_{z i}^{2} - S_{i} (S_{i} + 1) / 3 + {(E / D)}_{i} (S_{x i}^{2} - S_{y i}^{2})] {boldI}_{i} \cdot {boldP}_{i} \cdot {boldI}_{i} = (e Q V_{z i} / 12) [3 I_{z i}^{2} - I_{i} (I_{i} + 1) + η_{i} (I_{x i}^{2} - I_{y i}^{2})] H_{dip} = (β^{2} / 4 π {normalr}^{3}) [({boldg}_{1} \cdot {boldS}_{1}) \cdot ({boldg}_{2} \cdot {boldS}_{2}) - 3 ({boldg}_{1} \cdot {boldS}_{1} \cdot r) \cdot ({boldg}_{2} \cdot {boldS}_{2} \cdot r) / (

…”

Section: Methodsmentioning

confidence: 99%

Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures

et al. 2017

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“…The EPR simulation software ( SpinCount ) was written by one of the authors. 36 EPR samples were prepared under an inert atmosphere, sealed with a septum, and cooled to 77K unless otherwise mentioned. The sign of D for Mn(II)–OH and Mn(IV)–OH were determined from simulations of perpendicular-mode EPR spectra collected at variable temperatures.…”

Section: Methodsmentioning

confidence: 99%

Manganese–Hydroxido Complexes Supported by a Urea/Phosphinic Amide Tripodal Ligand

et al. 2018

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Hydrogen-bonds (H-bonds) within the secondary coordination sphere are often invoked as essential noncovalent interactions that lead to productive chemistry in metalloproteins. Incorporating these types of effects within synthetic systems has proven a challenge in molecular design that often requires the use of rigid organic scaffolds to support H-bond donors or acceptors. We describe the preparation and characterization of a new hybrid tripodal ligand ([H2pout]3–) that contains two mono-deprotonated urea groups and one phosphinic amide: the urea groups serve as H-bond donors while the phosphinic amide group serves as a single H-bond acceptor. The [H2pout]3– ligand was utilized to stabilizes a series of Mn–hydroxido complexes in which the oxidation state of the metal center ranges from 2+ to 4+. The molecular structure of MnIII–OH complex demonstrates that three intramolecular H-bonds involving the hydroxido ligand are formed. Additional evidence for the formation of intramolecular H-bonds was provided by vibrational spectroscopy in which the energy of the O–H vibration supports its assignment as an H-bond donor. The step-wise oxidation of [MnIIH2pout(OH)]2- to its higher oxidized analogs was further substantiated by electrochemical measurements and results from electronic absorbance and electron paramagnetic resonance spectroscopies. Our findings illustrate the utility of controlling both the primary and secondary coordination spheres to achieve structurally similar Mn–OH complexes with varying oxidation states.

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“…Isomer shifts are quoted relative to Fe metal at 298 K. All Mössbauer figures were prepared using SpinCount software. 19 …”

Section: Methodsmentioning

confidence: 99%

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

Komor¹,

Rivard

Fan

et al. 2016

J. Am. Chem. Soc.

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The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O2, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a 2-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spec product analysis of reactions under 18O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single turnover reaction between18O2–P and NH2-CAM is carried out in an 16O2 atmosphere, CAM nitro groups contain both 18O and 16O, suggesting that P can be re-formed during the reaction sequence. Such re-formation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM re-reduces the enzyme diiron cluster, allowing P to re-form upon O2 binding, while itself being oxidized to NO-CAM. Finally, the re-formed P oxidizes NO-CAM to CAM with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.

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Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes

Cited by 67 publications

References 23 publications

Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures

Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures

Manganese–Hydroxido Complexes Supported by a Urea/Phosphinic Amide Tripodal Ligand

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

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