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The Pennsylvania State University

The Graduate School

Eberly College of Science

MECHANISTIC STUDY OF MYO-INOSITOL OXYGENASE

A Dissertation in

Biochemistry, Microbiology and Molecular Biology

Yinghui Diao

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

August 2011

The dissertation of Yinghui Diao was reviewed and approved* by the following:

Joseph Martin Bollinger, Jr. Professor of Chemistry Professor of Biochemistry and Molecular Biology Dissertation Co-Adviser Co-Chair of Committee

Carsten Krebs Associate Professor of Chemistry Associate Professor of Biochemistry and Molecular Biology Dissertation Co-Adviser Co-Chair of Committee

Squire Booker Associate Professor of Chemistry Associate Professor of Biochemistry and Molecular Biology

John Golbeck Professor of Biochemistry and Biophysics Professor of Chemistry

Christopher House Associate Professor of Geosciences

Scott B. Selleck Professor and Head, Department of Biochemistry and Molecular Biology

*Signatures are on file in the Graduate School.

Abstract The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol

(cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to D-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and

O2, at nonheme diiron(II/III) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor.

Freeze-quench (FQ) technique allows to terminate reaction at desired time and trap reaction intermediate state, H, when it accumulates the highest. It is clear that H is diiron(II/III) complex according to Mössbauer spectra. Using chemical-quench-flow, we have distinguished different mechanisms by tracing the product formation kinetics.

The combined evidences favors the assignment of H as a MIOX(II/III)•product complex or an intermediate that breaks down to product upon quenching.

MIOX(II/III) is produced by comproportionation of the reactant MIOX(II/II) with a

MIOX(III/III) intermediate. UV-vis absorption, EPR and Mössbauer spectroscopies provide extensive evidence regarding how this process occurs.

The investigation on the reaction of MIOX(II/III) with substrate analogue L-epi- inosose-2 (MI-6-one) suggests dioxygenase activity of MIOX. Optical absorption,

EPR and Mössbauer spectroscopies show that the diiron (II/III) cluster of MIOX is perturbed upon addition of MI-6-one but maintains the oxidation state. The reaction of MIOX(II/III)•MI-6-one with O2 produces a product with +32 mass shift from MI-

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6-one. This isotope tracer experiments implies addition of 2 O-atoms and is reminiscent of the dioxygenases.

Table of Contents List of Figures ...... vii List of Tables ...... xiii List of Schemes ...... xiv Acknowledgements ...... xvi Chapter 1 ...... 1 Introduction to myo-Inositol Oxygenase (MIOX) ...... 1 MIOX Contains a Coupled Dinuclear Iron Cluster ...... 4 Indication of an Unconventional Mechanism ...... 5 Preparation of MIOX(II/III) and Characterization of MI Binding to it ...... 7

MIOX Uses the Mixed-Valent Fe2(II/III) Cluster as a Novel Cofactor for O2 Activation ...... 10 The Assignment of G as a (Superoxo)Diiron(III/III) Complex ...... 16 The Use of Metal Superoxo Intermediate for Substrate C-H Bond Activation ...... 18 The Three-Dimensional Structure of MIOX ...... 20 Possible Pathways for DG Production After C1-H Abstraction by G ...... 24 Chapter 2 ...... 30 Spectroscopic and Kinetic Approaches to Investigate the Nature of H ...... 30 Introduction ...... 32 Materials and Methods ...... 33 Results ...... 37 Discussion ...... 47 Chapter 3 ...... 50

Mechanism of the Reaction of the Fe2(II/II) Form of myo-Inositol Oxygenase [MIOX(II/II)] with O2 and H2O2 to Generate Active MIOX(II/III) ...... 50 Introduction ...... 52 Materials and Methods ...... 53 Results ...... 59 Discussion ...... 77 Chapter 4 ...... 83 Dioxygenase Activity of myo-Inositol Oxygenase from Mus musculus with the Substrate Analogue 2L-2,3,4,6/5-Pentahydroxycyclohexanone ...... 83

Introduction ...... 85 Materials and Methods ...... 91 Results ...... 98 Discussion ...... 113 Conclusion ...... 119 Chapter 5 ...... 120 Conclusions and Outlook ...... 120 Conclusions ...... 121 Outlook ...... 123 Bibliography ...... 127

List of Figures Figure 1-1 The binding of MI to MIOX(II/III) was monitored by optical absorption spectroscopy. The change in the spectrum is associated with addition of MI...... 8 Figure 1-2 Reaction of MIOX(II/III)•MI with limiting O2 monitored by SF absorption spectroscopy. This solution was mixed in a 1:1 volume ratio with air-saturated (at 5 °C in panel A and 23 °C in panel B) buffer. (A) The concentrations after mixing were 0.50 mM total MIOX, ~0.3 mM MIOX(II/III)MI, 0.15 mM O2, and 7.2 mM free MI. The spectra reflect the changes occurring between the reaction time of the first reliable spectrum (0.003 s) and reaction times of 0.010 (black), 0.027 (red), 0.070 (blue), 0.20 (green), 0.80 (purple), and 7.7 s (orange). (B) The concentrations after mixing were 0.50 mM total MIOX, ~0.3 mM MIOX(II/III)MI, 0.10 mM O2, and either 7.2 mM (black trace) or 25 mM (red trace) free MI...... 11 Figure 1-7 X-band EPR spectra of samples prepared by mixing MIOX(II/III) with different epimers of MI. The structure of individual epimer is color-coded and shown by the side of each corresponding spectrum...... 23 Figure 2-1 X-Band EPR spectra at 14 K and 20 mW of H (0.083-s FQ sample of the reaction of MIOX(II/III)•MI with O2, red), and of samples of MIOX(II/III)•DG (green) and MIOX(II/III)•L-gulonate (blue). Samples were prepared as described in Materials and Methods. Spectral parameters: microwave frequency, 9.45 GHz; modulation amplitude, 10 G; modulation frequency, 100 kHz; receiver gain, 1.25 × 104; time constant, 327 ms; points per spectrum, 1024; scan time, 168 s...... 38 Figure 2-2 Mössbauer spectra of MIOX. A solution of 1.49 mM MIOX(II/III)•MI reacts at 5 o o C with 2 equiv volume of O2 saturated buffer (5 C) and was quenched in 0.082 s. Experimental conditions are given in individual panel. A. 4.2-K/53-mT (top) and 120-K/zero-field (bottom) spectra of starting MIOX(II/III)•MI complex. Solid lines overlaid with the data are the contributions of MIOX(II/III)•MI (56% of the total intensity, blue) and MIOX(III/III)•MI (23% of the total intensity, green). B. Spectra of freeze-quenched sample enriched in H. The top spectra show raw date (hashed marks) and the contribution of MIOX(II/II)[•MI] (19% of the total intensity, red solid line), MIOX(III/III)•MI (24% of the total intensity, green solid line). Reference spectra of H were obtained by removing the 24 and 19% contributions of MIOX(III/III)•MI and MIOX(II/II)[•MI], respectively, and are shown at the bottom. The solid line overlaid with the 120-K/zero-field spectrum of H is a simulation according to the parameters from fit of three quadruple doublets in the text, and the dotted and solid lines plotted above the data are the contributions of the Fe(III) and Fe(II) sites, respectively. C. 120-k/zero-field of three independent freeze-quenched samples enriched in H. After removal of the contributions of MIOX(III/III)•MI (green solid lines) and MIOX(II/II)(•MI) (red solid lines) from raw spectra (top), reference spectra of H are shown in the bottom ...... 40 o Figure 2-3 Reaction of MIOX(II/III)•MI with limiting O2 at 5 C monitored by SF absorption spectroscopy in the absence (red) and presence (blue) of DG. The concentrations after mixing were 0.32 mM MIOX(II/III), 25 mM MI and 0.15 mM O2 for red trace and 0.32 mM MIOX(II/III), 25 mM MI, 0.4 mM DG and 0.15 mM O2...... 42 Figure 2-4 Enzymatic synthesis of D5-DG from D6-MI. A. LC-MS chromatogram of the 10 hours reaction mixture after removing protein. The reaction was carried out at 5 oC in a solution containing 2.3 mM MIOX(II/III), 40 mM D6-MI and 5 mM L-cysteine, exposed to the air for 10 hours. Single-ion monitoring at m/z 198 (M- for D5-DG) and

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185 (M- for D6-MI) was performed during elution. B. A standard curve of peak area of synthesized D5-DG (m/z 198, blue) versus D5-DG concentration was generated by identical analysis of samples with known H5-DG (m/z 193, red) concentrations and a concentration of 31.1 ± 0.3 mM was obtained...... 43 Figure 2-5 Kinetics of DG formation (black dots with error bars) in a single-turnover, chemical-quenched-flow experiment in which a solution containing 0.38 mM o MIOX(II/III), 100 mM MI and 0.1 mM D5-DG was mixed at 5 C with an equal volume of air-saturated (5 oC) buffer (giving 0.19 mM MIOX(II/III)•MI, 0.05 mM D5-DG and 0.11 mM O2 after mixing) and the reaction was quenched at desired times. Calculation of concentrations of DG is described in Materials and Methods. The error bars are the standard deviations for each reaction time in five independent trials. The dash line is a fit of the equation for an exponential increase and corresponds to a first- order rate constant of 11 s-1. The solid colored lines are simulations of the kinetic data according to the three possible cases outlined in the text [blue (overlapped with red) for the case in which H is a product complex, green for the case in which H is an intermediate that does not yield DG upon quenching and red for the case in which H is an intermediate that breaks down to DG upon quenching]...... 46 Figure 3-1 Mössbauer analysis of MIOX(II/II) activation by various amount of O2. A. Mössbauer spectra of MIOX samples recorded at 4.2-K in a 53-mT magnetic field applied parallel to the γ-beam. These samples were prepared as followed: an O2-free solution of 3 mM MIOX and 5.4 mM 57Fe(II) was rapidly mixed at 5 oC with a buffer containing different concentration of O2 to give final (O2/MIOX ratios of 0.032, 0.085, 0.14, 0.20 and 0.28), sealed in a reaction hose at 5 oC for 1 hour and added with 100 mM MI at the end in the anoxic chamber. The blue and green solid lines overlaid with the data are the contributions of MIOX(II/III)⦁MI and MIOX(III/III)⦁MI respectively. The color-coded arrows indicate the features of corresponding species. B. The fraction of different oxidation states of diiron cluster has been plotted with colored dots as the function of O2/MIOX ratio. Solid lines corresponding to different oxidation states are the simulation according to the model mechanism showed in Scheme 3-1. The error bars are the uncertainties of the Mössbauer analysis...... 60 Figure 3-2 The fraction of MIOX(II/III) in total diiron cluster determined by EPR analysis (closed circles) was plotted as the function of O2/MIOX ratio. Each sample was prepared by the rapid mixing of O2-free MIOX(II/II) with buffer containing different o concentration of O2 at different mixing ratio and subsequent incubation at 5 C for 1 hour. The only EPR sensitive species MIOX(II/III) was quantified with cooper standard and compared with the simulation according to model mechanism (Scheme 3-1)...... 62 Figure 3-3 Mössbauer analysis of MIOX(II/II) activation by various amount of H2O2. A. Mössbauer spectra of MIOX samples recorded at 4.2-K in a 53-mT magnetic field applied parallel to the γ-beam. These samples were prepared as followed: an O2-free solution of 3 mM MIOX and 4.8 mM 57Fe(II) was rapidly mixed with a buffer containing different concentration of H2O2 to give final H2O2/MIOX ratios of 0, 0.25, 0.50, 0.75, 1 and 2, sealed in a reaction hose at 5 oC for 1 hour and added with 100 mM MI at the end in the anoxic chamber. The blue and green solid lines overlaid with the data are the contributions of MIOX(II/III)⦁MI and MIOX(III/III)⦁MI respectively. B. The fraction of different oxidation states of diiron clusters has been plotted with colored dots as the function of H2O2/MIOX ratio. Solid lines viii

corresponding to different oxidation states are the simulations according to the model mechanism initiated with H2O2 showed in Scheme 3-1...... 64 Figure 3-4 Kinetics of the reaction of the MIOX(II/II) with excess O2. A. A sample of the MIOX(II/II) (0.3 mM) solution was mixed at 5 oC with an equal volume of buffer containing 0.95 mM O2. The concentration after mixing were 0.15 mM MIOX(II/II), o 0.48 mM O2. B. A same solution of MIOX(II/II) was mixed at 5 C with equal colume of buffer containing various concentration of O2. The final O2 concentrations are 0.24 mM (red), 0.48 mM (blue) and 0.71 mM (green). The solid lines are fits according to eq 1. The inset shows the fastest apparent first-order rate constants for the formation phase vs O2 concentration, which gives a second-order rate constant (slope) of 1.8 × 103 ± 20 M-1 s-1...... 65 Figure 3-5 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of MIOX(II/II) with excess O2. The anaerobic control sample (top spectrum) contained 2 mM MIOX, 3.6 mM 57Fe(II). The reaction of this reactant MIOX(II/II) with 2 o equiv volume of O2-saturated buffer (5 C) gave a final concentration of 0.6 mM MIOX(II/II) and 0.95 mM O2, and was rapidly freeze-quenched after desired times. A. The solid lines overlaid with the data are the sum of three contributions from MIOX(II/II), MIOX(II/III) and MIOX(III/III) using the reference spectra. The solid lines above the data are the contributions of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B. The kinetics of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). The solid lines are fits of the single exponential to the data. First-order rate constants of 2.0 ± 0.4 s-1 and 2.4 ± 1.0 s-1 were extracted from the fits to the MIOX(II/II) decay and the MIOX(III/III) formation, respectively. The error bars are the uncertainties of the Mössbauer analysis...... 66 Figure 3-6 Kinetics of the reaction of the MIOX(II/II) with excess H2O2. A sample of the MIOX(II/II) (0.3 mM) solution was mixed at 5 oC with an equal volume of buffer containing H2O2 (10 mM for panel A and 10 mM, 30 mM and 90 mM for panel B) to give a final concentration of 0.15 mM MIOX(II/II), 5 mM H2O2 in panel A and 5 mM (red), 15 mM (blue) and 30 mM (green) in panel B. B. The solid lines are fits according to eq 1. The inset shows the fasted apparent first-order rate constants for the formation phase vs H2O2 concentration, which gives a second-order rate constant (slope) of 2.1 × 103 ± 20 M-1 s-1...... 68 Figure 3-7 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of MIOX(II/II) with excess H2O2. The anaerobic control sample (top spectrum) contained 2 mM MIOX, 3.6 mM 57Fe(II). The reaction of this reactant MIOX(II/II) o was rapidly mixed at 5 C with equal volume of buffer containing 20 mM H2O2 gave a final concentration of 0.9 mM MIOX(II/II) and 10 mM H2O2, and was rapidly freeze-quenched after desired times. A. The solid lines overlaid with the data are the sum of three contributions from MIOX(II/II), MIOX(II/III) and MIOX(III/III) using the reference spectra. The solid lines above the data are the contributions of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B. The kinetics of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). The solid lines are fits of single exponential equations to the data and first-order rate constants of 44 ± 21 s-1 and 22 ± 8 s-1 were extracted for the decay of MIOX(II/II) and formation of MIOX(III/III), respectively. C. Overlay of Mössbauer spectra of the diiron(III) clusters in 0.02 s (green) and 10 min (blue) freeze-quenched samples. The spectrum of the 0.02 s sample was prepared by removing the 25% MIOX(II/III) and 39% MIOX(II/II) contributions from the raw spectrum of the 0.02 s sample, and the ix

spectrum of the 10 min sample was prepared by removing 25% MIOX(II/III). The solid lines are theoretical spectra simulated with parameters of the Fe species reported in the text...... 69 Figure 3-8 H2O2 concentration dependence of the absorbance at 490 nm of the quinone imine dye, generated by the HRP assay...... 71 Figure 3-9 A: 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of MIOX(II/II) with limiting O2. A reactant MIOX(II/II) was prepared by addition of 57 6.6 mM Fe(II) to 3.5 mM O2-free apo MIOX and frozen to make an anaerobic control sample. The reaction of this reactant MIOX(II/II) with equal volume of buffer containing 0.8 mM O2 gave a final concentration of 1.66 mM MIOX(II/II) and 0.4 mM O2, and was rapidly freeze-quenched after desired times. The solid lines overlaid with the data are the sum of theoretical reference spectra of different diiron forms with different contributions reported in the text, and the color-coded solid lines above the data are the contributions of the MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B: Kinetics of the different diiron clusters [MIOX(II/II) in red, MIOX(II/III) in blue and MIOX(III/III) in green] in the reaction. The solid lines are simulations of the kinetic data according to Scheme 3-2. Error bars are the uncertainties of Mössbauer analysis...... 73 Figure 3-10 Kinetics of reaction of MIOX(II/II) with limiting O2. In both SF and FQ experiment, a solution of 0.96 mM MIOX(II/II) was rapidly mixed at 5 oC with equal volume of air-saturated buffer (5 oC) to give a final concentration of 0.48 mM MIOX(II/II) and 0.11 mM O2. A. Reaction of MIOX(II/II) with limiting O2 monitored by SF absorption spectroscopy. The arrows indicate the changes of absorbance at ~ 600 nm and ~ 495 nm. B. X-Band EPR spectra of samples freeze- quenched at various reaction times during the limiting O2 reaction. The spectra were acquired at 10 K (nominal temperature on an Oxford cryostate controller) with a microwave frequency of 9.45 GHz, a microwave power of 20 mW, a modulation frequency of 100 kHz, a modulation amplitude of 10 G, a time constant of 327 ms, and a scan time of 167 s. The spectrum of a sample containing buffer alone was subtracted from all spectra to remove the feature intrinsic to the EPR cavity at 3380 G (g = 2.00). C. Kinetics of absorbance at 600 nm (dots, left y-axis) and of MIOX(II/III) (triangles, right y-axis) in the reaction. Calculation of the MIOX(II/III) concentrations from the intensities of the EPR features was described in Materials and Methods. The solid lines are simulations of the kinetic data according to Scheme 3-2...... 75 Figure 3-11 4.2-K/53-mT Mössbauer spectra of MIOX samples. A. A solution of 2.62 mM MIOX(II/II) was 1:1 mixed at 7 oC with same concentration of MIOX(III/III) for 1 hour to obtain a sample. The samples of starting MIOX(II/II), MIOX(III/III) and the mixture after 1 hour were all added with MI before freezing for analysis. B. 2.23 mM 57Fe MIOX(II/II) was 1:1 mixed at 7 oC with same concentration of 56Fe MIOX(III/III) and treated with 100 mM MI after desired times. The color-coded solid lines overlaid with the data are the contributions of the MIOX(II/II)(•MI) (red), MIOX(II/III)•MI (blue) and MIOX(III/III)•MI (green)...... 77 Figure 4-1 UV/visible absorption spectra of MIOX(II/III) in the absence and presence of MI- 6-one. A solution of 0.5 mM total MIOX enriched in MIOX(II/III) (0.24 mM, 1.6 eq. of Fe, red) was mixed with 73 mM MI-6-one to give a final 0.2 mM MIOX(II/III) and incubate for 2 hrs (at 7 °C, blue). The inset shows the change in the spectrum associated with addition of MI-6-one...... 99 x

Figure 4-2 Mass spectra depicting hydrogen exchange of MI-6-one in presence of MIOX(II/III). In a deuterium-enriched solution, 1 mM MI-6-one was incubated with either O2-free buffer or MIOX(II/III) (2.4 mM MIOX, 1.6 eq. of Fe, prepared as described in Materials and Methods, middle) for one hour. Heat-denatured protein was removed by centrifugation. The deuterated buffer of the supernatant was exchanged with protium-enriched buffer to wash out exchangeable deuteria. Representative relative intensity shows that MI-6-one (m/z = 177.2, green) gains one more mass (m/z = 178.2, blue) when mixing with MIOX(II/III)...... 100 Figure 4-3 EPR spectra of MIOX(II/III) in the absence and presence of MI-6-one. A solution of 0.5 mM total MIOX enriched in MIOX(II/III) (0.24 mM, 1.6 eq. of Fe, top) was mixed with 73 mM MI-6-one to give a final 0.2 mM MIOX(II/III) and incubate for 2 hours (at 7 °C, bottom)...... 101 Figure 4-4 Mössbauer spectra of MIOX(II/III). A sample of 57Fe-enriched MIOX(II/III) prepared by the comproportionation method described in Materials and Methods was recorded at 4.2 K in a 53-mT external magnetic field oriented parallel to the propagation direction of the γ-beam (top) and at 120 K without applied magnetic field (bottom). Solid lines above the data are the contributions of MIOX(II/II) (red, 29 ± 3%), MIOX(II/III) (blue, 60 ± 5%), and MIOX(III/III) (green, 11 ± 3%). The solid black lines overlaid with the experimental data (vertical bars) represent the added contribution of the three components...... 102 Figure 4-5 Mössbauer spectra of MIOX(II/III)•MI-6-one A sample of 57Fe subject to the comproportionation method described in methods and materials and then treated with 50 mM MI-6-one. A and B: 4.2-K/53-mT Mössbauer spectra of MIOX(II/III)•MI-6- one with parallel and perpendicular externally applied field (indicated on the top). C: 120-K/zero-field Mössbauer spectra of MIOX(II/III)•MI-6-one. Top spectra show raw data (hashed marks) and the contribution of MIOX(II/II)•MI-6-one (28% of the total intensity, solid line in red) and MIOX(III/III)•MI-6-one (9% of the total intensity, solid line in green). Removal of their contributions yields the reference spectra of MIOX(II/III)•MI-6-one (middle in A, bottom in B and C). The solid line overlaid with the data is a simulation according to the parameters from Table 1, and the solid and dotted lines plotted above the data are the contributions of the Fe(II) and Fe(III) sites, respectively...... 1043 Figure 4-6 Titration of 0.24 mM MIOX (II/III) with MI-6-one by EPR spectroscopy. The spectra shown correspond to 0 mM (red), 0.3 mM (blue), 0.9 mM (green), 2.7 mM (yellow), 24.3 mM (dark) and 72.9 mM (orange) MI-6-one. The points in the inset depict the occupancy of MIOX(II/III) as a function of MI-6-one concentration. The solid line is a fit of the quadratic equation for binding to the data, yielding Kd = 0.52 ± 0.09 mM...... 1056 Figure 4-7 Kinetics of absorbance at 720 nm after mixing (at 5 °C) of a solution enriched in MIOX(II/III) (0.2 mM) with an equal volume of an O2-free solution of MI-6-one to give a final MI-6-one concentration of 0.5 mM (o), 1.5 mM (□), 5 mM (◊), 15 mM (×) and 50 mM (+). The solid lines are fits of the equation for single first-order decay process to the data. The inset is a plot of the rate constant versus [MI-6-one] with a hyperbola fit, yielding Kd = 52 ± 4.4 mM...... 106 Figure 4-8 Kinetics of the reaction of the reaction of MIOX(II/III)•MI-6-one complex with excess O2. A sample of the MIOX(II/III)•MI-6-one complex (1 mM MIOX, 1.6 equiv of Fe, 50 mM MI-6-one) prepared as described in Materials and Methods, was mixed at 5 °C with an equal volume of the O2 saturated (at 5 °C) buffer to give a final 0.28 xi

mM MIOX(II/III), 25 mM MI-6-one, and 0.71 mM O2. The reaction was analyzed by both optical absorption (panel A) and X-band EPR (panel B) spectroscopies. (A) The spectra reflect the reaction times of 0.025 s (blue), 0.05 s (red), 0.10 s (green), 0.20 s (brown), 10 s (cyan) and 1000 s (black). (B) The samples were rapidly frozen at the indicated reaction times. The signal intensity of MIOX(II/III)•MI-6-one (obtained as described in Materials and Methods, black circles in panel C) is scaled for direct comparison to the absorbance change. Spectrometer conditions were as follows: temperature, 10 K; microwave frequency, 9.45 GHz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 10 G; time constant, 327 ms; scan time, 167 s. (C) The curve shows the kinetics of absorbance at 495 nm (red line) and 722 nm (blue line) which was directly compared to the EPR signal intensity. (D) Kinetics of reaction of the MIOX(II/III)•MI-6-one complex with excess O2. A solution of the MIOX(II/III)•MI-6-one complex [0.2 mM MIOX(II/III), 50 mM MI- o 6-one] was mixed at 5 C with an equal volume of an O2 soluiton. The final O2 concentrations are 0.18 (blue ○), 0.24 (red □), 0.36 mM (green ◊) and 0.71 (black Δ)

mM. The solid lines are fits according to eq 1. The inset shows kobs values for the decay phase of the reaction vs. O2 concentration. The plot gives second-order rate constant of (40 ± 2) × 103 M-1 s-1...... 1098 Figure 4-9 High performance liquid chromatogram mass spectrometry (HPLC-MS) analysis of the commercial MI-6-one and DS upon its incubation in 50 mM Bis-Tris-acetate, 10% (w:w) glycerol buffer (pH 6.0). MI-6-one (blue) and DS (red) were dissolved to 0.025 mM and 0.3 mM respectively in the buffer and analyzed by HPLC-MS, as described in the Material and Methods. The traces shown are of the intensity of negative-ion monitored at m/z 209 (M- for DS) and m/z 177 (M- for MI-6-one). Note that the peak centered at 23 min for m/z 209 is from the natural abundant 13C isotopologue of Bis-Tris (m/z = 208) in buffer which is dominated (50 mM) in the sample and were not seen in the standard DS sample dissolved in water...... 110 Figure 4-10 Mass spectra depicting intensities (A and B) and concentrations (C) of products 16 in the reaction of MIOX(II/III)•MI-6-one with either O2-free buffer (red), O2– 18 containing buffer (cyan and yellow), or O2-containing buffer (blue and green). In a sample, the MIOX(II/III)•MI-6-one complex prepared as described in Materials and 16 18 Methods was mixed with O2-free buffer (red), O2-saturated buffer or O2-saturated buffer subsequently followed by incubation at 5 °C for 30 min. Each oxygented solution was divided into two identical aliquots. An aliquot directly proceeded to heat-denaturing step (blue for 18O or cyan for 16O). A second aliquot solution was added with 0.15 mM DS as internal standard and followed by heat-denaturing (green for 18O or yellow for 16O). After removal of heat-denatured protein by centrifugation, the supernatant was subjected to the LC-MS for product analysis. A: The raw peak intensity of products from the reactions. B: The intensity of peak in anaerobic control sample was removed from that of each corresponding peak in the reaction samples. C: 18 16 The concentration of productsin the sample mixed with O2 (blue) or O2 (cyan) was obtained by comparing the peak intensity of product (m/z = 209-214) with that of 0.15 mM DS (m/z = 209) (see result section in text)...... 112

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List of Tables Table 1 Mössbauer Parameters of MIOX(II/III)•MI-6-one and MIOX(II/III)•MI (italicized values are parameters generated from 120-K/zero-field spectra) ...... 105

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List of Schemes Scheme 1-1 Reaction of O2 with the Fe2(II/II) cofactor of four dinuclear non-heme iron enzymes...... 2 Scheme 1-2 Catabolism pathway of inositol(poly)phosphates and phosphoinositides in human...... 3 Scheme 1-3 A mechanistic pathway for formation of 1-peroxy-MI...... 7 Scheme 1-4 Interconversion of three different oxidation states of the MIOX diiron cluster [(II/II), (II/III), and (III/III)] in the absence and presence of substrate, MI. Selected X- band EPR and 4.2-K/ 53-mT Mössbauer reference spectra are shown next to the respective structure...... 9 Scheme 1-5 Proposed mechanism of conversion of MI to DG initiated by the formally (superoxo)diiron (III/III) intermediate, G, upon abstraction of the hydrogen atom from C1. X-Band EPR spectra of the various states are shown underneath the structures...... 12 Scheme 1-6 Proposed mechanism involving iron superoxo complexes and subsequent H abstraction in IPNS (A), MIOX (B), HEPD (C) and HPPE (D)...... 19 Scheme 1-7 Possible pathways for conversion of G to the D-glucuronate. Pathway A involves O-O homolysis and rebound of a hydroxyl radical equivalent to the substrate radical. Pathway B involves Fe-O homolysis and rebound of a hydroperoxyl radical equivalent to the C1 radical. Pathway C involves C1 hydroperoxylation by addition of a second molecule of O2 to the C1 radical. Pathway D is proposed by Morokuma and highlighted with a hydroperoxo diiron(II/III) MI-1-one intermediate...... 26 Scheme 2-1 Kinetic mechanism used in the simulation of Figure 2-4. Two possible mechanisms were used in the simulation. The black one implies that H is an intermediate after G and proceed to form MIOX(II/III)•DG accompanying with C-C cleavage. On the right of mechanism, three traces are the simulations of production of H (blue) and DG under two different scenarios (green or red when H does or does not break down to DG upon quench, respectively). The red mechanism shows H is a product complex and directly release product and MIOX(II/III) in the rate-limiting step. Red trace on its right is the simulation of DG formation using this mechanism and parameters. The concentrations used for simulation are as same as chemical- quench-flow experiment...... 44 Scheme 2-2 A MIOX reaction pathway on the basis of DFT:MM study by Morokuma ...... 48 Scheme 3-1 One possible MIOX(II/II) activation mechanism in which H2O2 is formed in the first step...... 53 Scheme 3-2 Kinetic mechanism for activation of MIOX(II/II) used in the simulations of Figure 3-8 (solid lines)a ...... 78 Scheme 3-3 A possible tunnel between two molecules of MIOX and representative residues (cyan-red sticks) probably responsible for PCET. The distance is between two Fe1 sites of neighbor diiron centers (dark red balls). MI is shown as green-and-red-stick structure...... 81 Scheme 4-1 The natural substrate (MI) and substrate analogue (MI-6-one) reactions catalyzed by MIOX ...... 86 Scheme 4-2 Possible mechanisms for reaction MIOX(II/III)•MI with O2. Four possible reaction pathways (A - D) following cleavage of the C1-H bond by the formally superoxo-Fe2(III/III) intermediate, G, are depicted, which involve C1 hydroxylation coupled to peroxide O−O cleavage (pathway A), C1 hydroperoxylation by rebound

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of an HOO• radical (pathway B), C1 hydroperoxylation by addition of a second molecule of O2 to the C1 radical (pathway C) or electron transfer from C1 radical to the Fe site proposed by Morokuma (pathway D)...... 88 Scheme 4-3 Reactions catalyzed by the interdiol dioxygenases (A) and extradiol dioxygenases (B)...... 114 Scheme 4-4 Possible mechanisms for the reaction of MIOX(II/III)•MI-6-one with O2...... 116

Acknowledgements First of all, I want to express my deeply-felt thank to both of my advisors, Prof. Marty Bollinger and Prof. Carsten Krebs for giving me the opportunities to work on such exciting and intellectually stimulating projects. I will always be thankful for their encouragements and inspiring guidance throughout my Ph.D study. Their enthusiasm and persistence for science turn time-consuming, difficult research into a joyful journey and always help me overcome difficulties. My appreciation also goes to other faculty members, Prof. Squire Booker, Prof. John Golbeck and Prof. Christopher House, for serving on my committee. I owe special thanks to Dr. Gang Xing, who started this project from 2003. He started to train me to become a biochemist from every aspect on the first day when I joined this project. I learned from him that how performing every single step carefully could finally make breakthrough. I would like to take this opportunity to thank my colleagues in Bollinger/Krebs group. Eric Barr was my freeze-quench/chemical-quench partner for many days and nights. Lee Hoffart did a large amount of Mössbauer analysis for this project. Denise Corner synthesized the specific labeled substrates and provided insightful discussion on NMR analysis. Ning Li also help me with freeze-quench experiments in the last few months of my Ph.D study. Megan Matthews created artistic MIOX structure pictures. I also want to thank all the members in Bollinger/Krebs group for their help and support. I will never forget your encouragements and those thoughtful notes from my defense practice. I owe my parents too much for not being with them in past eight years especially when they encountered the biggest difficulty. Their tremendous support and unconditional love has been, and will always be, inspiring me. My beloved son, Andrew Shi, is the sunshine in my life. He lets me know how extremely happy a mother can be. Last but not least, I thank my husband, Kebin Shi. His support and understanding always come to me immediately whenever I face problems or difficulties. He lets me in State College, a small town far far away from our home country, experience endless faith and love.

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Chapter 1 Introduction to myo-Inositol Oxygenase (MIOX)

The dinuclear non-heme iron enzymes serve diverse functions in important reactions including hydroxylation in methane monooxygenase (MMO), 1 e- oxidation in ribonucleotide reductase (RNR), and desaturation in Δ9 desaturase (Δ9D) (1-5).

Among these reactions oxygen activation is an intensely intriguing topic because of biomedical or industrial potential (2, 6-8). Dioxygen is a reactant for most enzymes in this family. The uncatalyzed reactions of O2 (S = 1) with organic substrates (S = 0) are thermodynamically favorable but kinetically slow because they are spin-forbidden and the one-electron reduction potential of O2 is modest. All dinuclear non-heme

proteins and enzymes mentioned above have in common that the reduced, Fe2(II/II) form reacts with O2 to generate a peroxo-Fe2(III/III) intermediate as the first step for

Scheme 1-1 Reaction of O2 with the Fe2(II/II) cofactor of four dinuclear non-heme iron enzymes. diverse reaction outcomes (Scheme1-1). An exception is myo-inositol oxygenase

(MIOX) which uses an unconventional dinuclear non-heme-iron cofactor to activate

O2 and initiate a complicated glycol-cleaving, four-electron-oxidation reaction (9-11).

MIOX catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to D-glucuronate (DG), the initial step in the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides (12). Subsequent transformations convert DG to xylitol (12, 13)

(Scheme 1-2). Accumulated evidence indicates that both depletion of intracellular MI and accumulation of polyols, including xylitol, are associated with various diabetic complications such as nephropathy, retinopathy, neuropathy, and cataract (14).

Recent work by the Kanwar group suggested that MIOX modulates various downstream pathways affected by high-glucose ambience and may play a role in the

Scheme 1-2 Catabolism pathway of inositol(poly)phosphates and phosphoinositides in human.

pathobiology of tubulointerstitium (substance in the renal tubules and interstitial tubules) in diabetic nephropathy (15). MIOX could be a target for treatment of diabetic complications, and an understanding of its catalytic mechanism could thus be extraordinarily valuable. However in the ~50 years between the time MIOX was first described and shown to require iron (16) and the start of my Ph. D. research in 2005, little insight into its structure and catalytic mechanism has subsequently emerged, at least partially because of the difficulty to obtain highly active MIOX in high purity and yield. The isolation of the genes encoding MIOX orthologues from several organisms (17) and the overexpression of MIOX in E. coli (18) overcame this challenge.

MIOX Contains a Coupled Dinuclear Iron Cluster

The dependence of MIOX activity on iron had been known for over 50 years (16).

However, the stoichiometry of iron in the protein had not been clarified until we launched over investigation in 2005. A simple metal dependent activity assay experiment first indicated that the maximal activity was obtained when two equivalents of iron were reconstituted into the apo MIOX (unpublished data by Xing

G., Barr E. W., Krebs C., and Bollinger J. M., Jr.). A Mössbauer spectrum of a sample prepared by mixing iron-free MIOX with Fe(II), in the absence of O2 then treated with an excess of either O2 or H2O2 exhibited a quadrupole doublet (at 4.2 K and 53 mT) with isomer shift and quadrupole splitting parameters characteristic of high-spin Fe(III) ions with nitrogen and oxygen coordination. The absence of magnetic hyperfine splitting suggested that the complex might have an integer-spin 4

ground state, while the spectrum acquired in a strong external magnetic field (6 T) showed that the complex has a diamagnetic (S = 0) ground state, which could simply be rationalized by antiferromagnetic (AF) coupling of two high-spin Fe(III) ions in a

Fe2(III/III) cluster (9). Our Mössbauer study provided the first direct evidence for

MIOX as a non-heme diiron enzyme.

Indication of an Unconventional Mechanism

Typically, the enzymes from the family of dinuclear non-heme-iron oxidases and

oxygenases activate O2 at the reduced, Fe2(II/II) form of the cofactor to generate high- valent intermediates that are sufficiently potent to cleave unactivated C-H bonds (1,

2). At the end of the oxidation sequence, the Fe2(III/III) form is produced (1, 3, 4). In order to return to the initial Fe2(II/II) active state and complete the catalytic cycle, re-

reduction of the Fe2(III/III) to Fe2(II/II) by a reductase protein using NAD(P)H is required (3, 4). Therefore, with two electrons provided by the nicotinamide co- substrate, no more than two electrons can be extracted from the substrate in a single cycle. MIOX, on the other hand, carries out a four-electron oxidation of MI without any additional co-substrate or reductant. It was hard to understand how MIOX could activate oxygen and catalyze the reaction by a mechanism similar to those of conventional non-heme diiron enzymes. An early experiment promptly gave us an informative hint. A solution containing apo enzyme, 2 equiv. Fe(II) and saturating MI was mixed with limiting O2. If MIOX would work as other enzymes that activate O2 at the Fe2(II/II) form of diiron cofactors, then one would expect that one DG per O2 would be produced and, importantly, that the reduced form would be regenerated 5

after the consumption of the limiting reactant (O2). Unexpectedly, much less DG was produced and the enzyme was converted to the mixed-valent, Fe2(II/III) state with a stoichiometry of 1.1 ± 0.2 per O2. The accumulation of the mixed-valent form under single-turnover conditions strongly indicated that the II/III state might be the active enzyme form. A radiometric assay for DG production in the reactions of MIOX with the diiron cofactor in different oxidation states with limiting O2 also suggested that

MIOX (II/III) was more likely the active enzyme state. A solution of either MIOX

(II/II) or MIOX (II/III) was mixed with 14C-labeled MI, followed by the addition of limiting O2. Incubation at ambient temperature for a few minutes allowed the reaction to reach completion. Anion-exchange chromatography separated unreacted [14C]MI from [14C]DG product and fractions were subjected to liquid scintillation counting to quantify the substrate and product. A stoichiometry of 0.8 DG/O2 in the reaction of

14 MIOX(II/III)34[ C]MI with limiting O2 was detected, similar to the theoretical value of 1. By comparison, only 0.5 DG/O2 was generated by the reduced enzyme state under the same condition. The formation of DG from the reduced enzyme would not be difficult to understand after we studied the activation of MIOX(II/II) by O2

(discussed in Chapter 3). The Fe2(II/II) can react with O2 to generate Fe2(II/III) in a yield of 4 Fe2(II/III)/ per O2. It is almost impossible to have zero amount of Fe2(II/III) in a solution of MIOX(II/II) due to the contaminating O2 in the anoxic chamber.

Therefore, this small amount of DG generated in the reaction initiated with the reduced enzyme state is more likely from the catalytically active form, MIOX(II/III).

In a mechanism proposed by Hamilton and co-workers (19), L-myo-inosose-1 was suggested as a probable intermediate. In this mechanism, MI reacts with and oxidized form of the enzyme to give a 1-keto intermediate (L-myo-inosose-1), and O2 is then converted by the reduced enzyme to a peroxide that attacks the carbonyl group of the intermediate (Scheme 1-3). We ruled out his hypothesis for a couple of reasons. First,

Mössbauer spectra showed that the binding of MI to MIOX(III/III) did not change its oxidation state. Second, the binding of MI to MIOX(III/III) was too slow to be competent for the catalysis. Third, it has been reported that H2O2, which converts

MIOX(II/II) to MIOX(III/III), inactivates the enzyme (20) and that catalase therefore

Scheme 1-3 A mechanistic pathway for formation of 1-peroxy-MI. protects it (19). Fourth and the most importantly, MIOX(III/III) has very low activity but can be fully reactivated by treatment with reductants such as L-Cys or ascorbate.

Preparation of MIOX(II/III) and Characterization of MI Binding to it

An antiferromagnetically (AF)-coupled, high-spin Fe2(II/III) cluster can be formed either by slow diffusion of O2 (g) into a solution containing apo MIOX, 2 equiv Fe(II) and reductant (L-cysteine or ascorbate), or by reduction of MIOX(III/III) by L- cysteine. Both methods give a similar yield of 60%. A third method of enriching

MIOX(II/III), which involves comproportionation of MIOX(II/II) and MIOX(III/III), will be discussed in detail in Chapter 3. Addition of MI to MIOX(II/III) changes the

-1 -1 absorption rapidly (kon = 260 ± 20 M s ) giving a positive feature at ~ 390 nm and a negative feature at ~ 495 nm, corresponding to the formation of MIOX(II/III)⦁MI and the decay of substrate-free mixed-valent enzyme respectively (Figure 1-1). Stopped- flow (SF) UV-vis spectroscopy showed that the binding of MI to MIOX(II/III) is kinetically competent to be on the catalytic pathway, with the association rate constant of 260 ± 20 M-1s-1

-1 -1 exceeding kcat/Km (70 M s ) by almost four- fold. MI binding also perturbs the Mössbauer and electron paramagnetic resonance (EPR) Figure 1-1 The binding of MI to MIOX(II/III) was monitored by spectra of the mixed-valent diiron cluster optical absorption spectroscopy. (Scheme 1-4). The 4.2-K/53-mT Mössbauer The change in the spectrum is spectrum of MIOX(II/III) exhibits broad, poorly associated with addition of MI. resolved absorption from -6 mm s-1 to +6 mm s-1. Together with the 120-K/zero-field spectrum, it suggests conformational heterogeneity in MIOX(II/III). In contrast to the poorly resolved features of MIOX(II/III), MIOX(II/III)·MI yields well-resolved

Mössbauer features at -1.9 mm s-1 and 3.7 mm s-1 arising from the Fe(II) site and at -

3.8 mm s-1 and +4.5 mm s-1 arising from the Fe(III) site (the peak positions refer to the most intense outer lines of the two magnetic subspectra; Scheme 1-4). At 120-K and zero-field, each subspectrum collapses to a quadrupole doublet with parameters typical of distinct high-spin Fe(II) and high-spin Fe(III) sites. This Mössbauer analysis confirmed that the procedures for preparing MIOX(II/III) give similar yield of ~60% MIOX(II/III) though the concentration of MIOX(II/II) and MIOX(III/III) varies depending on the method used. MI-free MIOX(II/III) displays a broad, axial

EPR spectrum with g-values of 1.95, 1.66 and 1.66, which is detectable only at

Scheme 1-4 Interconversion of three different oxidation states of the MIOX diiron cluster [(II/II), (II/III), and (III/III)] in the absence and presence of substrate, MI. Selected X-band EPR and 4.2-K/ 53-mT Mössbauer reference spectra are shown next to the respective structure.

relatively low temperature (< 8 K) and high microwave power (20 mW). The substrate bound form gives rise to a much sharper spectrum with g-values of 1.95,

1.81 and 1.81, which is readily observable at higher temperature and lower microwave power (e.g., 20 K and 0.10 mW). The signals are typical of AF-coupled, valence-localized, high-spin Fe2(II/III) clusters, consistent with our Mössbauer analysis. Both clusters exhibit S = 1/2 EPR signals with a gaverage of < 2 (Scheme 1-4).

From our spectroscopic characterization of mouse MIOX(II/III) in the presence and absence of MI, we speculated that the substrate coordinates directly to the diiron cluster, possibly via an alkoxide bridge. To test for it, Q-band 2H-ENDOR spectra of the complexes prepared either with MI substituted with deuterium at all six carbons

[MIOX(II/III) •D6-MI] or with unlabeled MI [MIOX(II/III) •H6-MI] were acquired at magnetic fields spanning the axial EPR signal (21). According to a structural model analysis, the 2H ENDOR analysis shows MI indeed binds to the mixed-valent diiron center of MIOX directly via a bridging alkoxide. However, a crystal structure published soon afterward suggested a different coordination mode. In this mode, MI binds in bidentate mode through its C1 and C6 hydroxyls. (See later).

MIOX Uses the Mixed-Valent Fe2(II/III) Cluster as a Novel Cofactor for O2 Activation

The hallmarks of catalysis first appeared in a reaction of mixed-valent enzyme

o substrate complex [MIOX(II/III) •MI] with limiting O2 at 5 C. Time-dependent absorption difference spectra revealed features opposite to the difference spectrum for

MI binding. A negative feature at ~ 390 nm, indicating loss of the substrate complex,

developed and decayed, implying that the MIOX(II/III) •MI complex was consumed, and a positive feature at ~ 495 nm developed soon after, suggesting that MI-free

MIOX(II/III) was then produced. Within ~ 8 s after mixing, difference absorption decayed almost completely, suggesting that the starting MIOX(II/III) •MI complex was regenerated after the limiting O2 was consumed (Figure 1-2). The cycle was fast enough to account for the steady-state turnover number (~ 0.3 s-1) under the same reaction conditions. Decay of the positive 495 nm peak was

Figure 1-2 Reaction of MIOX(II/III)•MI with limiting O2 monitored by SF absorption spectroscopy. This solution was mixed in a 1:1 volume ratio with air-saturated (at 5 °C in panel A and 23 °C in panel B) buffer. (A) The concentrations after mixing were 0.50 mM total MIOX, ~0.3 mM MIOX(II/III)MI, 0.15 mM O2, and 7.2 mM free MI. The spectra reflect the changes occurring between the reaction time of the first reliable spectrum (0.003 s) and reaction times of 0.010 (black), 0.027 (red), 0.070 (blue), 0.20 (green), 0.80 (purple), and 7.7 s (orange). (B) The concentrations after mixing were 0.50 mM total

MIOX, ~0.3 mM MIOX(II/III)MI, 0.10 mM O2, and either 7.2 mM (black trace) or 25 mM (red trace) free MI. accelerated by increased [MI], and the maximum absorption was consequently diminished at higher [MI], proving that this decay reflects re-binding of MI to the substrate-free form and that MIOX(II/III) did indeed accumulate during the reaction,

consistent with the hypothesis that turnover occurred (Figure 1-2). The temporal separation between development of the negative 390 nm and positive 495 nm features suggested that at least one intermediate state accumulated between MIOX(II/III) •MI and MIOX(II/III).

EPR spectra of samples frozen through the course of the single-turnover confirmed that MIOX(II/III) •MI reacts rapidly with O2 and is subsequently regenerated. The kinetics of MIOX(II/III)•MI are very similar to what is extracted from absorbance at

390 nm in the SF absorption experiments. More importantly, the samples provided direct evidence for an intermediate state. The transient decay of the g = (1.95, 1.81,

1.81) signal of MIOX(II/III) •MI was accompanied by development of a new, rhombic g = (1.92,1.76,1.54) signal (Scheme 1-5). Formation and decay of the new signal were shown to track precisely with decay and re-development of the

MIOX(II/III) •MI SF signal. These features represented an accumulating intermediate, as anticipated from the SF data. Part of my project has been to investigate the nature

II/III II/III•MI G H Scheme 1-5 Proposed mechanism of conversion of MI to DG initiated by the formally (superoxo)diiron (III/III) intermediate, G, upon abstraction of the hydrogen atom from C1. X-Band EPR spectra of the various states are shown underneath the structures.

of this intermediate, designated H. We initially considered two possibilities. One possibility is that H is a product complex. In this scenario, product release would be the rate-limiting step. However, a simple EPR experiment performed by addition of either the product, DG, or its C6-aldehyde-reduced analogue, L-gulonate (which cannot cyclize, and thus should mimic well the acyclic form of DG produced in the

MIOX active site), directly to MIOX (II/III) disfavored this possibility because the sample was found to exhibit a rhombic EPR signal distinct from that of H (Scheme 1-

4). H could still be the MIOX(II/III) •DG complex, but with the DG and /or cofactor in an altered state that can be accessed only through turnover. Indeed, in previous studies on taurine:α-ketoglutamate dioxygenase, a mononuclear nonheme-iron enzyme, we were unable to generate a known product complex by addition of products to the resting enzyme (22). The second possibility is that H could be a true chemical intermediate. In this case, an additional state, the MIOX(II/III) •DG product complex would be required after H in the minimal kinetic mechanism for the reaction.

Chapter 2 presents spectroscopic and kinetic evidence and suggests that H is more likely a product complex or an intermediate that breaks down to the product upon quenching.

Substrate Deuterium Kinetic Isotope Effect (2H-KIE) Allows Detection of the C-H-Cleaving Intermediate, G

2 SF absorption with the use of deuterium-labeled substrate [1,2,3,4,5,6-[ H]6-MI (D6-

MI)] allowed demonstration of a significant 2H-KIE on this reaction. The trace of 390

nm representing substrate bound MIOX(II/III) for the D6-MI has an initial rising phase preceding the decay and redevelopment phases. By comparison, the trace for

H6-MI exhibits only an early decay phase and similar redevelopment phase. These data suggest that an additional intermediate that absorbs more intensely than

MIOX(II/III) •MI accumulates exclusively (or to a much greater extent) in the D6-MI reaction. Faster decay in the H6-MI reaction disfavors the accumulation of this intermediate. Kinetic difference spectra show that the positive difference in absorption associated with the presumptive new intermediate maximizes around 435 nm. The significant 2H-KIE on the decay of this intermediate strongly suggests that this species is involved in C-H (D) cleavage. Confirmed and detected by freeze- quench (FQ) EPR spectroscopy, this new intermediate was designated G in the reaction of MIOX(II/III) •D6-MI with O2. A rhombic S = 1/2 signal with effective g values of 2.05, 1.98, and 1.90 develops rapidly and decays completely within a few hundred milliseconds as the previously detected intermediate, H, develops. Kinetic Figure 1-3. Kinetics of MIOX(II/III)•D6-MI (black), G (red) data established that G is a precursor of H and H (blue) determined from the FQ and the hydrogen-abstracting species based EPR experiment. Concentrations are expressed in terms of the fraction of on the observations that: (1) it accumulates the initial [MIOX(II/III)D6-MI]. The to a much lesser extent with H6-MI; and (2) solid lines are simulations according to Scheme 1-3. the lesser accumulation of G results from a

greater accumulation of H. Due to negligible accumulation of G in the H6-

MI reaction, only a rough estimation of intrinsic 2H-KIE (8-16) could be obtained. One interesting property of G is the reversibility of its formation from

MIOX(II/III)•MI and O2, which is Figure 1-4 Photolytic decay of G at 77 K. indicated by the biphasic decay of Top: The spectrum of the sample enriched in G. Bottom: The spectrum was acquired MIOX(II/III)•MI and the coincidence of after subsequent exposure of the same the slow phase with decay of its sample to laboratory fluorescent light for 40 min in liquid nitrogen. successor, G (Figure 1-3). In addition, comparison of the spectroscopic data of the reactions carried out with H6-MI and D6-

MI at 0.5 oC rather than 5 oC clearly shows higher intensity of MIOX(II/III)•MI in the

D6-MI reaction at early time points (20 ms and 43 ms), which strongly corroborates the reversibility of G formation. Another feature of G is its photolytic lability to ambient laboratory light at cryogenic temperatures (77 K) which was used to quantify

G in H6-MI and D6-MI samples from thedifference of spectra before and after light exposure (Figure 1-4).

A kinetic mechanism comprising MIOX(II/III), MIOX(II/III)•MI, G, H and

MIOX(II/III)•DG was proposed and accurately accounted for both the kinetic data

-1 from the D6-MI reaction and the steady-state kcat (0.7 s ) (Scheme 1-4). This mechanism predicts a 2H-KIE on steady-state turnover of close to unity because the

o decay of H is almost totally rate-limiting for the turnover of even D6-MI (at 5 C).

The examination of the steady-state velocities at 5 oC by me [by the orcinol colorimetric assay; see ref (23)] gave a 2H-KIE of 1.35 ± 0.35, which is consistent with the prediction of scheme 1-4.

The Assignment of G as a (Superoxo)Diiron(III/III) Complex

G is the intermediate upon O2 addition to MIOX(II/III)•MI, and its rhombic g ≈ 2

EPR signal establishes that it has an S = 1/2 ground state. The most simple interpretation is that G is formally a (superoxo)diiron(III/III) complex formed upon addition of O2 to the Fe(II) site of the reactant complex. In principle, there are other possible states: [(hydro)peroxo]diiron(III/IV) complex formed by two-electron oxidative addition of O2 to the diiron(II/III) cluster, or a more advanced state formed by addition of O2 and subsequent O-O cleavage with further oxidation of the Fe ions

[e.g. di-(µ-oxo)diiron(IV/V)]. The high-valent oxidation states and/or a cleaved O-O bond seem unlikely. To our knowledge, formation of Fe(IV) enzyme intermediates has thus far been seen only in conjunction with O-O bond cleavage. It is not obvious that reduction of O2 by two electrons to a peroxide would be sufficiently exergonic to drive oxidation of an Fe ion of the diiron(II/III) reactant to the high-valent state. The reversibility of G formation strongly favors formulations in which the O-O bond remains intact. Reversibility in O-O cleavage is, to our knowledge, unprecedented for diiron complexes, although it has been demonstrated for an inorganic dicopper complex (24). Moreover, Que and coworkers recently prepared and characterized by resonance Raman spectroscopy an inorganic (superoxo)diiron(II/III) complex from a 16

diiron(II/II) precursor and O2 (25). A superoxide ligand to a diiron(III/III) cluster, as proposed for G, should be at least as stable with respect to further reduction to peroxide as the diiron(II/III)-coordinated superoxide of the model complex.

Formulations with either terminal or bridging superoxide coordination can be envisioned for G. Such a complex would contain three paramagnetic centers, two high-spin Fe(III) ions (S = 5/2) and the superoxide radical anion (S =1/2). The experimentally observed S = 1/2 ground state can be rationalized by assuming antiferromagnetic coupling between the two Fe(III) ions and between the superoxide and the Fe(III) site(s) to which it binds. For coordination to only one Fe(III) (FeA) site, exchange coupling between the superoxide and the other Fe(III) (FeB) should be much smaller and negligible. As a consequence, the spin of FeA would align antiparallel to those of the superoxide and FeB, resulting in an S = 1/2 ground state.

Alternatively, a bridging superoxide would yield two similar antiferromagnetic

Fe(III)-superoxide exchange interactions, resulting in spin-frustrated cluster. Using I

= 1/2 57Fe nuclei hyperfine coupling to the electron spin, the two modes are expected to be distinguishabledue to different magnitude of the hyperfine interactions. The photolysis procedure was used to experimentally resolve the spectra of G prepared from 56Fe- and 57Fe-containing MIOX(II/III)·MI. For the superoxide-bridged complex, the spin projection factors of two Fe(III) sites should be identical and relatively small. For superoxide coordination only to FeA, the coefficients would be bigger. Simulations of the EPR spectra, starting with that of the 56Fe-labeled complex and applying isotropic hyperfine couplings of 13 G and 25 G for the two (presumed)

high-spin Fe(III) sites to this theoretical spectrum to reproduce the experimental spectrum of the 57Fe-labeled complex, were carried out. The magnitude of the overall broadening of the EPR spectrum is incompatible with the small couplings predicted for the bridged complex but compatible with the couplings predicted for the terminal complex. We therefore favor assignment of G as a (superoxo)diiron(III/III) complex with either end-on (η1) or side-on (η2) coordination of the superoxide to a single

Fe(III). Very recently, Morokuma and coworkers investigated the structure of G by means of density functional theory (DFT) and ONION quantum mechanical/molecular mechanical (QM/MM) approaches (26). A side-on coordination geometry and an S = 1/2 spin state were assigned to the ground state of the intermediate, in which the two iron sites are antiferromagnetically coupled and the

Fe1 and the superoxide site are ferromagnetically coupled. Moreover, population and spin density analyses showed that, in G, charge transfer from Fe1 to O2 is not complete, thus G is better described as a “partial” (superoxo)diiron(III/III) species.

Nevertheless G is capable of abstracting hydrogen from a C−H bond, which supports our proposed mechanism and calculated 2H-KIE.

The Use of Metal Superoxo Intermediate for Substrate C-H Bond Activation

Rather than high-valent iron-oxo (ferryl) complex, a set of non-heme-iron oxygenases and oxidases use iron superoxo complexes to abstract hydrogen from their substrates. These enzymes include MIOX (10, 11), isopenicillin N synthase

(IPNS) (27), 2-hydroxyethyl phosphonate dioxygenase (HEPD) (28, 29),

Scheme 1-6 Proposed mechanism involving iron superoxo complexes and subsequent H abstraction in IPNS (A), MIOX (B), HEPD (C) and HPPE (D).

CloR (30) and possibly 2-hydroxypropylphonate epoxidase (HPPE) (Scheme 1-6)

(31-33). Many of them catalyze four-electron oxidations of the substrates by a single equivalent of dioxygen without the consumption of external reductants or cofactors

(34). Iron superoxo (“pre-ferryl”) complexes are generated in catalytic pathways of these enzymes to abstract hydrogen from their substrates, which allows these enzymes to obtain the electrons needed to cleave the O−O bond of O2, providing the thermodynamic driving force for their difficult oxidation reactions (34). MIOX to date is one of two enzymes in which the proposed iron superoxo complex was detected, characterized and demonstrated as the H-abstracting intermediate. The other enzyme utilizing superoxo complex to do H-abstraction is IPNS (Esta Tamanaha, unpublished data). Besides iron, other metal, copper is also thought to be used to form superoxo complex in two uncoupled dicopper monooxygenases: dopamine β-

monooxygenase (DβM) and peptidylglycine α-amidating monooxygenase (PHM) to cleave C-H bonds (35-37). In addition, Nam and co-workers reported an inorganic

Cr(III)-superoxo complex with the tetradentate ligand 14-TMC (14-TMC = 1,4,8,11- tetramethyl-1,4,8,11-tetraacyclotetradecane), which is capable of abstracting a H atom from alkylaromatic substrates (38) These novel intermediates significantly expand the range of important transformations of aliphatic carbon compounds involving cleavage of C-H bonds and confer remarkable catalytic versatility.

The Three-Dimensional Structure of MIOX

A major breakthrough in the understanding of MIOX is the three-dimensional structure of the mouse enzyme determined by X-ray crystallography. The Baker group reported for the first time the crystal structure of mouse MIOX, in complex with MI (39). The structure

[presumably the fully oxidized state

MIOX(III/III)•MI because of the time Figure 1-5 Ribbon diagram of the mouse required for crystal growth, the aerobic MIOX fold showing helices 4, 5 and 8 (in pink) comprised by the conserved conditions, and the absence of L-cysteine HD domain structure that contributes from the crystallization solution] four of the six Fe ligands and helices 7 and 8 (in cyan) containing the remaining revealed the overall protein fold, two unique histidine ligands that identified the iron ligands (Figure 1-5), complete the Fe2 site. and confirmed the direct coordination of MI to the cofactor, albeit in a mode distinct from the µ-O1 mode that we had speculated (21) (Figure 1-6). Sequence analysis had

indicated that MIOX is structurally

unrelated to the conventional diiron

oxygenase/oxidase proteins and this

difference was confirmed by the structure.

Similar to RNR and MMO, the diiron site of

Figure 1-6 Active site of mouse MIOX MIOX is deeply buried between two adapted from the structure solved by antiparallel helix pairs. However, MIOX Baker and coworkers with superoxide modeled into the Fe1 site representing lacks the EXXH ligand motif conserved in the superoxide-diiron(III/III) those evolutionarily related protein such as intermediate, G. The distance from the RNR, MMO and Δ9 desaturase (40, 41). terminal oxygen atom of superoxide to C1 of MI is 1.92 Å. Interestingly, one of the two iron sub-sites

(Fe1) belongs to a structurally distinct family, the HD-domain superfamily, which is a conserved divalent-metal-binding structure and first recognized by Aravind and Koonin in metal-dependent phosphohydrolases (42). HD domains contribute the strictly conserved metal ligands, two histidines (H) and two aspartates (D), for a single metal site. Figure 1-7. Superposition of the Similarly in MIOX, these ligands are structure of mouse and human contained in α4, α5 and α8. For the enzymes (in grey and blue, respectively). second iron sub-site (Fe2, the site that

coordinates the substrate), two histidine residues (H194 in α6 and H220 in α7) serve as the metal ligands, although α6 and α7 are not part of the HD domain. Thus, it appears that MIOX evolved by elaboration of an HD cassette with two additional ligands that provide the coordination environment for a second iron sub-site (Fe2), allowing for formation of a coupled dinuclear cluster. More recently, a higher- resolution structure of N-terminally truncated form of human MIOX revealed a very similar geometry of the diiron site (Figure 1-7) (43). Given the fact that (i) crystals were grown in the presence of 1mM L-cysteine and 2 mM tris-

(carboxyethyl)phospine and (ii) L-cysteine can reduce inactive MIOX(III/III) to

MIOX(II/III), it is possible that this structure may represent the mixed-valent form of the partially active, truncated human MIOX. If so, the similarity of the active site geometries would suggest that the details deduced by Baker are essentially correct for the functional II/III form.

One key implication from the three-dimensional structures is the substrate-binding mode. We had proposed the µ-O1 substrate-binding mode on the basis of chemical considerations and the ENDOR data (21). However, the Baker structure showed MI coordinated only to Fe2 in an η2-(O1, O6) chelating mode with the substrate in its most stable chair conformation (O2 axial, O1 and O3-6 equatorial). They proposed that substrate coordinated to single Fe2 site (presumably in the +3 oxidation state) and Fe1 should be the ferrous site in the active mixed-valent form responsible for O2 activation. One striking difference between the structure of the mouse MIOX and the truncated human enzyme is the bound substrate species. myo-Inosose-1 (the 2-

electron-oxidized 1-ketone form of MI, abbreviates MI-1-one) was assigned by the author as the bound species, despite the fact that only MI was added in the crystallization. Interestingly, myo-inosose-1 was proposed as an intermediate in the catalytic pathway by Hamilton and co-workers (see above) (19).

The X-ray structure provides further information on the substrate binding interactions. The VGD motif (residues 140-142 in mouse MIOX) is conserved in all

MIOX sequences and is involved in a hydrogen bond network with O4 of MI, suggesting that the configuration of the C4 hydroxyl group is important for MI binding. An experiment for defining the minimum structural unit that must be present in an MI analogue for binding and catalysis were performed with the use of EPR spectroscopy (Figure 1-8, Yinghui Diao, unpublished data). The mixing of either L-

+ myo-inositol

+ L-chiro-inositol

+ scyllo-inosiol

+ D-chiro-inositol

+ epi-inositol

+ neo-inositol

Figure 1-8 X-band EPR spectra of samples prepared by mixing MIOX(II/III) with different epimers of MI. The structure of individual epimer is color-coded and shown by the side of each corresponding spectrum.

chiro-inositol (C1 epimer) or epi-inositol (C4 or C6 epimer respectively) with

MIOX(II/III) generated much weaker EPR signals than those of MIOX(II/III)•MI, whereas the EPR spectra of diiron(II/III) cluster after binding of either scyllo-inositol

(C2 epimer), D-chiro-inositol (C3 epimer) or neo-inositol (C5 epimer) showed significant intensity. It suggested that the configurations of C1, C6 and C4 are critical for substrate binding and the configurations of C2, C3 and C5 are less critical, which is qualitatively consistent with the binding mode implied by the x-ray structure.

Possible Pathways for DG Production After C1-H Abstraction by G

After abstraction of the C1–bonded H-atom by the uncoordinated O–atom of G, subsequent oxidative steps lead to formation of a new C1–O bond and cleavage of the

O–O and substrate O6–H and C1–C6 bonds. For these steps, two classes of mechanisms were previously proposed (Scheme 1-7 pathways A and B) (10). In pathway A, designated the “hydroxylation” pathway, the (hydroperoxo)diiron(III/III) intermediate decays by attack of the C1 radical on the distal O-atom of the hydroperoxide, leading simultaneously to hydroxylation of C1 and cleavage of the O–

O bond. The resultant substrate species would be a coordinated gem-diol(ate), and the cofactor would be in the III/IV oxidation state. The electron-deficient cofactor would then serve as an “electron sink” to permit cleavage of the C1–C6 bond. This pathway is analogous to one of the two mechanisms proposed for HEPD, in which the substrate radical attack the hydroperoxide generating a ferryl intermediate and a hemiacetal (29). In pathway B, which we designate the “hydroperoxylation” pathway, formation of the new C1–O bond precedes the bond-cleavage steps. Based on the X- 24

ray structure, this pathway has the rather stringent geometric requirement for the Fe- coordinated O-atom of the O2 unit to come into sufficient proximity to C1 to be transferred after the other O-atom abstracts the C1-H. Alternatively, it is conceivable that the proton is translocated from the distal to the proximal O-atom of the Fe(III)- hydroperoxide moiety, followed by “hydroperoxylation” via attack of the distal O- atom on C1 and Fe-Oproximal homolysis. The intermediate in this pathway, (1- hydroperoxy)-myo-inositol, was first envisaged by Hamilton and co-workers in the

1980s, who cited chemical precedent that it would break down to DG (19). A third pathway, which has not been previously discussed, invokes addition of a second molecule of O2 to the C1 radical (pathway C in Scheme 1-7), thus suggesting involvement of both a “cofactor oxygen” to abstract the C1 hydrogen and a “substrate oxygen” to effect the oxidative C-C cleavage. Some analogies of it include the oxygenation step known to occur in lipoxygenases (44, 45), an molecular oxygen attack on the radical at the N-prenyl moiety proposed to occur in the α-ketoglutarate- dependent oxygenase, FtmOx1 (46) and O2-mediated cleavage of glycyl radical enzymes (47). Recently this pathway has taken on renewed interest on the basis of our study of the MIOX reaction with the 6-ketone analogue of MI, L-epi-2-inosose

(MI-6-one).The oxidation of MI-6-one mediated by MIOX(II/III) appears to be related mechanistically to the well-studied reactions of ring-cleaving catechol dioxygenases, in which O2 is thought to add concertedly to the Fe cofactor and the

III substrate to form a bridging Fe −O−O−Csubstrate peroxide that produces a diacid product (48). The study of the oxidation of MI-6-one mediated by MIOX(II/III) will

Scheme 1-7 Possible pathways for conversion of G to the D-glucuronate. Pathway A involves O-O homolysis and rebound of a hydroxyl radical equivalent to the substrate radical. Pathway B involves Fe-O homolysis and rebound of a hydroperoxyl radical equivalent to the C1 radical. Pathway C involves C1 hydroperoxylation by addition of a second molecule of O2 to the C1 radical. Pathway D is proposed by Morokuma and highlighted with a hydroperoxo diiron(II/III) MI-1-one intermediate.

be discussed in detail in Chapter 4. A fourth pathway (pathway D in Scheme 1-7) was proposed by Morokuma on the basis of the DFT and ONIOM (DFR:MM) study (26).

Their analysis of the electronic structure of the hydroperoxo-Fe2(III/III)•C1-substrate radical intermediate (the state after H-atom abstraction by G) suggests that this state is better described as a hydroperoxo-Fe2(II/III)•myo-inosose-1 complex, which is formally obtained by inner-sphere one-electron transfer from the ketyl radical to Fe2.

Morokuma proposed that this state represents H. The key step in the Morokuma mechanism entails nucleophilic attack of the peroxide moiety on the carbonyl C1- atom. Homolytic O−O bond cleavage was predicted to be the rate-limiting step of the reaction and did not exhibit 2H-KIE, which is consistent with our experimental observation t. Morokuma’s calculations are significant to that the 2H-KIE on the steady-state turnover was close to unity (10). Interestingly, the role of hydroxide ligand is not only to bridge the two iron ions but also to serve as a catalytic base in the reaction.

The contents of subsequent chapters are briefly summarized. Chapter 2 presents the results of attempts to determine the nature of intermediate H. Mössbauer studies

suggest that H is a valence-localized Fe2(II/III) species. Chemical quench kinetics favor the assignment of H as the MIOX(II/III)•product complex.

Chapter 3 presents characterization of the activation of the MIOX(II/II) to

MIOX(II/III) by O2 and H2O2. A MIOX(II/III)/O2 stoichiometry study shows that 1 equivalent O2 can produce 4 equivalents of MIOX(II/III). Mössbauer, EPR and

optical absorption spectroscopies combined with kinetics studies indicate an activation mechanism in which MIOX(III/III) is generated as an early intermediate and subsequently converts to the mixed-valent state by slow comproportionation with

MIOX(II/II).

Chapter 4 presents characterization of an alternative dioxygenase reaction that

MIOX catalyzes with the substrate analogue, L-epi-inosose-2 (MI-6-one). The reaction of MIOX(II/III)•MI-6-one with O2 generates a product with +32 mass shift compared to the substrate analogue, MI-6-one, which indicates that two O atoms are incorporated into the product. The combination of different spectroscopies demonstrates that the MIOX(II/III) is perturbed upon binding of MI-6-one without changing its oxidation state. FQ EPR and SF kinetic study imply a cyclic and productive reaction. A deuterium exchange study by mass spectrometry indicates that a tautomerization and the formation of an enediol take place when MIOX(II/III) is

18 present. Finally an O2 labeling study reveals that the product contain 68% incorporation of a single atom of 18O, and 32% incorporation of a second atom of 18O, which confirms the dioxygenase outcome, yet that mechanistic discussion would be in the chapter 4.

In chapter 5, an outlook and future direction will be discussed. The characterization of C1-H-bond cleaving intermediate G is crucial for understanding the mechanism of the MIOX reaction. A combination of kinetics and multiple spectroscopic analyses will provide insight into the nature of G. With the use of different substrate analogues,

the reaction could be impeded and allow intermediates to accumulate, permitting the intermediate to be further characterized.

Chapter 2 Spectroscopic and Kinetic Approaches to Investigate the Nature of H

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to catalyze the four-electron oxidation of the myo-inositol (MI) to

D-glucuronate ((DG). A formally(superoxo)diiron(III/III) intermediate, G, was detected with the use of deuterium-labeled substrate and assigned as the C1−H- cleaving species. H, the successor of G, was detected in the reaction with unlabeled substrate and displays a g = (1.94, 1.76, 1.54) EPR signal. The decay of H is the rate- limiting step in the catalytic cycle. Mössbauer spectroscopy provides a strong evidence to reveal that H is a valence-localized diiron(II/III) cluster. By use of the chemical-quench-flow and LC-MS, the kinetics of DG production relative to those of the spectroscopically detected intermediate was defined to investigate the nature of H.

Comparison of the experimental kinetics of DG production with the simulations under different assumptions for H indicates that the intermediate H is either a product complex or an intermediate that breaks down to DG upon the denaturing chemical

(acid) quench.

Introduction

Characterization of the reaction of MIOX(II/III)•MI with O2 by a combination of

SF absorption and FQ EPR revealed the presence of two EPR-active reaction intermediates with S = 1/2 ground states. The first intermediate in the reaction sequence, termed G, was identified as the C1-H-cleaving species on the basis of the large 2H kinetic isotope effect (KIE) of approximately 8-15 on its decay. G is best formulated as a (superoxo)diiron(III/III) complex with terminal coordination of superoxide to one of the Fe sites, presumably Fe1 based on the X-ray structure (39).

Decay of G leads to generation of a near-stoichiometric amount of the second intermediate, H, which exhibits an S = 1/2 ground state with a broad, rhombic EPR signal [g = (1.94, 1.76, 1.54)]. H decays in the rate-limiting step of the catalytic cycle back to MIOX(II/III) which rebounds MI to complete the cycle. The structure of H and its placement in the reaction mechanism are only poorly understood. Two scenarios are possible. H could be the product complex, in which case the slow step of the reaction (decay of H) would be dissociation of product, or alternatively H would be a reaction intermediate between G and MIOX(II/III)•DG, in which case decay of H to the MIOX(II/III)•DG product complex is the slow step and subsequent steps leading to the regeneration of the reactant MIOX(II/III)•MI complex are faster.

Previous EPR-spectroscopic experiments (summarized below for completeness) showed that the EPR features of H are distinct from those of the Fe2(II/III) complex generated by addition of the product, DG, to MIOX(II/III), perhaps arguing for the second scenario. Additional FQ-Mössbauer and chemical-quench LC/MS

experiments described in this chapter focus on unraveling the nature of H in more detail.

Materials and Methods

Freeze-Quench (FQ) EPR Experiments.

The apparatus and procedures for preparation of FQ EPR samples have been described previously (49). MIOX(II/III)•MI was prepared by the comproportionation method (described in chapter 3) followed by addition of MI (final concentrations of

0.85 mM MIOX, 1.23 mM Fe, and 100 mM MI). The MIOX(II/III)•MI solution was

o o mixed at 5 C with 2 equiv volumes of O2-saturated (5 C) MIOX buffer [50 mM

Bis-Tris-acetate (pH 6.0) and 10% (w/w) glycerol, giving 1.4 mM O2] to give final concentrations of 0.13 mM MIOX(II/III)•MI and 0.95 mM O2, and the reaction solution was freeze-quenched after 0.020 s to obtain the highest yield of H according to the minimal kinetic mechanism (9). The EPR spectrometer has been described previously (50). Spectral parameters are given in the appropriate figure legend.

Preparation of EPR Samples of MIOX(II/III) Complex with either D-glucuronate or

L-gulonate.

The C6-aldehyde-reduced analogue of DG, L-gulonate, was prepared by base hydrolysis of the corresponding γ-lactone (Sigma-Aldrich). A solution of 0.65 M L- gulonic acid γ-lactone was mixed with 0.7 molar equiv NaOH and reacted at 58 oC for

1 hour. Progress of the reaction was monitored by mass spectrometry with electrospray ionization in the negative ion mode. The mobile phase and mass 33

spectrometer were described previously (11). When > 95% L-gulonic acid γ-lactone

(m/z = 177) converted to L-gulonate (m/z = 195) by peak height, the reaction was

assumed to be complete. The solution was directly used for preparation of EPR

samples. Preparation of all complexes was carried out in an MBraun anoxic chamber.

Details of the sample preparation are given in the figure legends.

Preparation of FQ Mössbauer Samples.

The apparatus and procedures for preparation of FQ Mössbauer samples have been described previously (49). The MIOX(II/III)•MI solution [containing 3.10 mM MIOX,

5.33 mM 57Fe, 100 mM MI and 1.49 mM enriched in diiron(II/III)•MI form] was

o o mixed at 5 C with 2 equiv volume of O2-saturated buffer (5 C, giving final concentrations of 0.50 mM MIOX(II/III)•MI and 0.95 mM O2), and the reaction solution was freeze-quenched after 0.082 s [H accumulates to a maximum amount after this reaction time, based on previous results (11)].

EPR and Mössbauer Spectroscopy.

The spectrometers have been described previously (50). Specific conditions are

given in the appropriate figure legend. Simulation of Mössbauer spectra was carried

out using WMOSS (WEB Research, Edina, MN).

Chemical Quench (CQ) Experiment for Kinetics of Product Formation.

The CQ experiment was carried out in an MBraun anoxic chamber. Preparation of

CQ samples is similar to preparation of samples by the FQ method. The essential difference is that termination of the reaction is achieved by spraying the reaction solution into a mixture of 20% acetic acid and 80% isopropyl alcohol (v/v) as the quench solution. In a single-turnover reaction, a solution containing 0.38 mM

o MIOX(II/III), 100 mM MI and 0.1 mM D5-DG as internal standard was mixed at 5 C with an equal volume of air-saturated (5 oC) buffer (giving final concentrations of

0.19 mM MIOX(II/III)•MI, 0.05 mM D5-DG and 0.11 mM O2) and quenched at the desired reaction time with 10 equiv volume of quench solution. By comparison of theoretical (simulated) kinetic traces to the data, a “quench time” (the time required for denaturing of the protein by quench solution) of 0.015 s could be estimated. This is longer than typical quench times reported in acid quench enzymological experiments but we believe, consistent with the method by which the reaction and quench solution were mixed by the spraying the reaction mixture to an eppendorf tube containing acid quench solution. The quoted reaction time for each sample is the sum of the known transit time through the reaction hose and the estimated “quench time” of 0.015 s. The quenched reaction solution was taken out of the anoxic chamber.

Next, the precipitated protein was removed by centrifugation, and the supernatant

(mixture of quench solution and water) was dried in vacuo using a Savant SpeedVac®

Concentrator (Thermo Scientific) for 10 hours. The sample was dissolved in 100 µL mobile phase (11 mM NH4Ac/56% acetonitrile/44% water, pH = 7.2) and the pH was adjusted to 7.0 by addition of 2 µL 14.5 M NH4OH. The mobile phase and NH4OH

were removed in vacuo by using a speed-vac. The dry sample was dissolved in 70 µL mobile phase for LC-MS analysis.

Synthesis of Internal Standard D5-DG for Chemical Quench Quantification

The synthesis of D5-DG is through the enzymatic reaction catalyzed by MIOX. The enzyme reaction was carried out in the standard buffer [50 mM Bis-Tris-acetate (pH

6.0) and 10% (w/w) glycerol]. A 500 µL solution containing 2.26 mM MIOX(II/III),

40 mM D6-MI (CDN Isotopes, Pointe-Claire, Quebec, Canada) and 5 mM L-cysteine was exposed to the air at 5 oC for 5 hours. Addition of another 500 µL 2.26 mM

MIOX(II/III) completed the reaction. The solution was centrifuged through a

Microcon YM-3 centrifugal concentrator (Millipore, Bedford, MA) to remove protein.

The filtered solution was diluted with 0.05% triethylamine/50% methanol/50% water mobile phase in variable concentrations and dilutions were added with a standard solution of unlabeled DG (referred to in the following as H5-DG) of known concentration (40 mM). A 10 µL aliquot of each diluted solution was injected onto a

PRP-X300 anion exclusion column (250 mm × 4.1 mm, 7µm particle size; Hamilton,

Reno, NV) in the mobile phase described above. The column was developed

(isocratically) at a flow rate of 0.1 mL/min. Single-ion monitoring at m/z 198 (M- for

D5-DG) and 193 (M- for H5-DG) were performed during elution, and the area under the peak (centered at 14 min) was determined. A standard curve of peak area at m/z

198 versus D5-DG concentrations was generated by identical analysis of peak area at m/z 193 of known H5-DG concentrations.

Quantification of DG Production from Chemical Quench Experiments.

A 10 µL aliquot of each treated chemical quench sample was injected onto a 150 mm × 2.1 mm ZIC-HILIC (hydrophilic interaction liquid chromatography) silica- based column (SeQuant) with zwitterionic stationary phase

+ - [−CH2N (CH3)2(CH2)3SO3 or sulfobetaine]. The column was developed (isocratically) at a flow rate of 0.1 mL/min in 11 mM NH4AC/56% acetonitrile/44% water /pH 7.2 mobile phase. The mass spectrometer was described previously (11). Single-ion monitoring at m/z 198 (M- for added D5-DG) and 193 (M- for produced H5-DG) were performed during elution, and the areas under the peaks (centered at 2.7 min) were determined. The concentration of H5-DG generated in the single-turnover reaction was determined ratiometrically by comparison to the area of the D5-DG peak.

Kinetic Simulations.

Kinetic simulations were performed with KinTekSim (KinTek Corp., Austin, TX).

The mechanism and parameters are presented in Results.

Results

EPR Spectra Indicate that H is Different from the Complex Generated by Addition of

DG to MIOX(II/III)

In the reaction of the MIOX(II/III)•MI complex with excess O2 (0.95 mM), after

0.083 s the features associated with H develop (g = 1.94, 1.76 and ~1.54). The large deviation of the g-values from ge suggests the presence of low-lying excited electronic states. Previous EPR-spectroscopic results, in which the 4.2-K EPR reference spectrum of H was compared to that of a sample of MIOX(II/III) treated with 100 mM DG and found to be distinct, suggested that H is unlikely to be the

MIOX(II/III)•DG product complex (11). A caveat for this conclusion is the fact that

DG in solution exists primarily as the cyclic hemiacetal, while DG produced by

MIOX(II/III)-catalyzed oxidation of MI is in its acyclic form. The analogue of the product, in which the C6-aldehyde group is reduced to the alcohol, L-gulonate, Figure 2-1 X-Band EPR spectra at 14 K cannot cyclize and may therefore be a and 20 mW of H (0.083-s FQ sample of better model for DG in the catalytically the reaction of MIOX(II/III)•MI with O2, red), and of samples of MIOX(II/III)•DG relevant MIOX(II/III)•DG complex. The (green) and MIOX(II/III)•L-gulonate spectrum of the MIOX(II/III)•L-gulonate (blue). Samples were prepared as described in Materials and Methods. complex also exhibits a rhombic EPR Spectral parameters: microwave spectrum distinct from that of H (Figure 2- frequency, 9.45 GHz; modulation amplitude, 10 G; modulation frequency, 1). Again, this result is not definitive proof 100 kHz; receiver gain, 1.25 × 104; time that H is not the MIOX(II/III)•DG product constant, 327 ms; points per spectrum, 1024; scan time, 168 s. complex, because L-gulonate is

chemically distinct from DG.

Mössbauer Spectroscopic Characterization of H

To further characterize H, we used FQ Mössbauer spectroscopy. Analysis of the

4.2-K/53-mT and 120-K/zero-field spectra of the reactant solution reveals that the sample contains 56% of MIOX(II/III)•MI, 21% of MIOX(II/II)[•MI], and 23% of

MIOX(III/III)•MI (Figure 2-2A). From previous studies, it is known that under the reaction conditions employed in this experiment MIOX(II/III)•MI is nearly quantitatively (> 95%) converted to H (11). Thus, H should contribute 55% of the total intensity of the spectrum of the 0.082 s sample. Moreover, the oxidation of

MIOX(II/II)[•MI] by O2 needs to be taken into account. Experiments described in

Chapter 3 reveal that the reaction of MIOX(II/II) with O2 is slow, compared to the reaction of MIOX(II/III)•MI with O2, and it is estimated that 2% of MIOX(II/II)[•MI] has been oxidized to MIOX(III/III) under the reaction conditions. Therefore, we expect the 0.082 s sample to contain 56% H, 1% MIOX(II/III)•MI, 19% MIOX(II/II)

(•MI), and 24% MIOX(III/III)•MI. Removal of the contribution of the latter two components from the raw data (red and green lines, respectively in Figure 2-2B, top) yields the reference spectra of H (Figure 2-2B, bottom). The 120-K/zero-field reference spectrum of H provides direct evidence that it is a valence-localized cluster with distinct Fe sites that exhibit Mössbauer parameters typical of high-spin Fe(III) and high-spin Fe(II). The spectrum can be fitted with two quadrupole doublets of equal intensity and parameters (1) = 0.47 mm/s, ΔEQ(1) = 0.85 mm/s, (2) = 1.20

A B

C 120 K 0 T

Figure 2-2 Mössbauer spectra of MIOX. A solution of 1.49 mM MIOX(II/III)•MI reacts o o at 5 C with 2 equiv volume of O2 saturated buffer (5 C) and was quenched in 0.082 s. Experimental conditions are given in individual panel. A. 4.2-K/53-mT (top) and 120- K/zero-field (bottom) spectra of starting MIOX(II/III)•MI complex. Solid lines overlaid with the data are the contributions of MIOX(II/III)•MI (56% of the total intensity, blue) and MIOX(III/III)•MI (23% of the total intensity, green). B. Spectra of freeze-quenched sample enriched in H. The top spectra show raw date (hashed marks) and the contribution of MIOX(II/II)[•MI] (19% of the total intensity, red solid line), MIOX(III/III)•MI (24% of the total intensity, green solid line). Reference spectra of H were obtained by removing the 24 and 19% contributions of MIOX(III/III)•MI and MIOX(II/II)[•MI], respectively, and are shown at the bottom. The solid line overlaid with the 120-K/zero-field spectrum of H is a simulation according to the parameters from fit of three quadruple doublets in the text, and the dotted and solid lines plotted above the data are the contributions of the Fe(III) and Fe(II) sites, respectively. C. 120-k/zero-field of three independent freeze- quenched samples enriched in H. After removal of the contributions of MIOX(III/III)•MI (green solid lines) and MIOX(II/II)(•MI) (red solid lines) from raw spectra (top), reference spectra of H are shown in the bottom .

mm/s, and ΔEQ(2) = 2.43 mm/s. The line width of the two quadrupole doublets is rather large [Γ(1) = 0.41 mm/s and Γ(2) = 0.27 mm/s] suggesting that H exhibits some heterogeneity. The high-energy line of Fe(III) in the reference spectrum indeed reveals two partially resolved peaks. The 0.082 s sample may include multiple states along the catalytic pathway. The spectrum can be fitted with a revised three quadruple doublets with the parameters δ(1) = 0.46 mm/s, ΔEQ(1)= 0.70 mm/s, δ(2) = 0.46 mm/s, ΔEQ(2) = 1.22 mm/s, δ(3) = 1.21 mm/s and ΔEQ = 2.42 mm/s. Three independent freeze-quenched samples enriched in H were analyzed at 120-K/zero- field and compared (Figure 2-2C). Although the contribution of the Fe(II) site from

MIOX(II/II)(•MI) are not identical in the three samples (more in the left two samples than the right sample) due to the different method used for making the

MIOX(II/III)•MI complex, we have confirmed that the predominant MIOX(II/III)•MI form was generated in similar yield (65-70%) and retained identical catalytic activity

(see Chapter 3). After removal of the contributions from the MIOX(II/II)(•MI) and

MIOX(III/III)•MI (red and blue lines in Figure 2-2C, top), three reference spectra of

H were generated and are shown in Figure 2-2C bottom. They reveal very similar features that correspond to a valence-localized (II/III) cluster with distinct Fe sites.

The high energy lines of Fe(III) in all three samples display partially resolved or broad peaks that suggest heterogeneity of H. The 4.2-K/53-mT reference spectrum of

H (Figure 2-2B) exhibits, as expected, paramagnetically split features, of which the magnitudes of the splittings are consistent with the presence of a valence-localized

Fe2(II/III) cluster. Because the spectral simulation of such a cluster depends on more

than 20 parameters, we have not attempted to simulate the spectrum. Thus, FQ

Mössbauer spectroscopy provides the mechanistically valuable information that H harbors a valence-localized Fe2(II/III) cluster.

Synthesis of Internal Standard D5-DG for Chemical Quench Quantification

In the chemical quench experiment, to minimize errors caused by variability in sample composition rising from variation in the relative volumes of reaction and quench solutions mixed, a known concentration of an isotope labeled product, D5-DG, was added to the reactant complex MIOX(II/III)•MI as an internal standard for MS quantification. Minimal D5-DG (0.05 mM) was needed to satisfy the MS detection limit under the assay conditions. To ensure that addition of labeled product would not inhibit the reaction, a single-turnover reaction of an solution containing

MIOX(II/III)•MI and minimal D5-DG with Figure 2-3 Reaction of limiting O2 was carried out. This reaction MIOX(II/III)•MI with limiting O2 at 5 o revealed no kinetic difference (Figure 2-3). It C monitored by SF absorption spectroscopy in the absence (red) and is easy to understand because competing presence (blue) of DG. The with saturated substrate, product inhibition is concentrations after mixing were 0.32 mM MIOX(II/III), 25 mM MI and almost negligible. D5-DG is the product of 0.15 mM O2 for red trace and 0.32 mM MIOX reaction with isotope labeled MIOX(II/III), 25 mM MI, 0.4 mM DG

and 0.15 mM O2. substrate, D6-MI. In 10 hours, the reaction

was determined to be complete on the basis of the mass spectrometry result. Figure 2-

4A implies that all D6-MI was converted to D5-DG. The concentration of synthesized

D5-DG (m/z =198) in the stock solution was determined by comparison to the known concentration of H5-DG (m/z = 193) with -5 mass shift, to be a value of 31.1 ± 0.3 mM (Figure 2-4B).

A B

Figure 2-4 Enzymatic synthesis of D5-DG from D6-MI. A. LC-MS chromatogram of the 10 hours reaction mixture after removing protein. The reaction was carried out at 5 oC in a solution containing 2.3 mM MIOX(II/III), 40 mM D6-MI and 5 mM L-cysteine, exposed to the air for 10 hours. Single-ion monitoring at m/z 198 (M- for D5-DG) and 185 (M- for

D6-MI) was performed during elution. B. A standard curve of peak area of synthesized

D5-DG (m/z 198, blue) versus D5-DG concentration was generated by identical analysis of samples with known H5-DG (m/z 193, red) concentrations and a concentration of 31.1 ± 0.3 mM was obtained.

Implication of Nature of H by Chemical Quench

To obtain further insight into the nature of H, we carried out chemical-quenched-flow experiments analyzed by LC-MS to define the kinetics of DG production relative to

those of the spectroscopically detected intermediate. Scheme 2-1 depicts kinetic simulations [employing the experimental reaction conditions and kinetic parameters determined previously (10, 11)] that illustrate the rationale that, if H is assumed to be the product complex, then the formation of DG would be expected to correlate temporally with the formation of H (top, right-hand kinetic trace). By contrast, if H is an intermediate preceding the product complex, then DG formation should correlate in time with the decay of H (bottom, right-hand simulation, red trace). The caveat for the latter case is that certain possible partially processed MI-derived species might break down to DG upon the denaturing chemical (acid) quench. For example, if H

H is MIOX(II/III)•product

H is Intermediate

Scheme 2-1 Kinetic mechanism used in the simulation of Figure 2-4. Two possible mechanisms were used in the simulation. The black one implies that H is an intermediate after G and proceed to form MIOX(II/III)•DG accompanying with C-C cleavage. On the right of mechanism, three traces are the simulations of production of H (blue) and DG under two different scenarios (green or red when H does or does not break down to DG upon quench, respectively). The red mechanism shows H is a product complex and directly release product and MIOX(II/III) in the rate-limiting step. Red trace on its right is the simulation of DG formation using this mechanism and parameters. The concentrations used for simulation are as same as chemical-quench-flow experiment.

were to contain the 1-hydroperoxy-MI intermediate proposed by Hamilton, et al. (19), one would anticipate that this species might yield DG upon quenching, thus obscuring the fact that H contains an MI-derived intermediate other than the product. The observation that addition of DG to MIOX(II/III) elicits an EPR spectrum different from that of H (Figure 2-1) would seem to weigh in favor of the former possibility

(that H is s true intermediate rather than the product complex), but we considered it is still possible that H could be a complex of MIOX(II/III) with acyclic DG, whereas

DG in solution, having formed the hemiacetal, binds to the enzyme in this form to elicit a different EPR spectrum.

The chemical-quenched-flow experiments were carried out at an initial concentration of O2 (0.11 mM) sufficiently less than the initial concentration of

MIOX(II/III)•MI (0.19 mM) to ensure that only a single-turnover could occur.

Acid/organic denaturant quenching was employed and estimated to have a “quench time” of 0.015 s. The kinetics of DG formation averaged over 5 independent trials are shown in Figure 2-5 (circular points with error bars representing the standard deviations). We infer that the non-zero concentration of DG results from the scavenging of O2 in the anoxic chamber by MIOX(II/III)•MI. The total change in

[DG] with time is similar to that expected from the limiting [O2] of 0.11 mM.

The kinetics of DG production indicate that H is either the MIOX(II/III)•DG complex (blue trace in Figure 2-5) or an intermediate containing an MI-derived species that breaks down to the product upon quenching (red trace in Figure 2-5).

Figure 2-5 Kinetics of DG formation (black dots with error bars) in a single-turnover, chemical-quenched-flow experiment in which a solution containing 0.38 mM o MIOX(II/III), 100 mM MI and 0.1 mM D5-DG was mixed at 5 C with an equal volume o of air-saturated (5 C) buffer (giving 0.19 mM MIOX(II/III)•MI, 0.05 mM D5-DG and

0.11 mM O2 after mixing) and the reaction was quenched at desired times. Calculation of concentrations of DG is described in Materials and Methods. The error bars are the standard deviations for each reaction time in five independent trials. The dash line is a fit of the equation for an exponential increase and corresponds to a first-order rate constant of 11 s-1. The solid colored lines are simulations of the kinetic data according to the three possible cases outlined in the text [blue (overlapped with red) for the case in which H is a product complex, green for the case in which H is an intermediate that does not yield DG upon quenching and red for the case in which H is an intermediate that breaks down to DG upon quenching].

Effect of solvent viscosity on the reaction

In principle, the effect of solvent viscosity on the kinetics of DG production in the steady-state might be used to distinguish whether H is the MIOX(II/III)•DG product complex or an intermediate preceding MIOX(II/III)•DG. Because decay of H appears to be the overall rate-limiting step in the reaction, the former case predicts that increasing solvent viscosity, expected to slow the physical step of product

dissociation, should slow the reaction, as was shown for example for the mononuclear non-heme-iron enzyme taurine:-ketoglutarate dioxygenase (TauD) (22). By contrast, in the latter case, decay of H would involve a chemical step (see Scheme 2-2 in

Discussion) which one would expect to be less sensitive to solvent viscosity.

Unfortunately, when glycerol was used as viscosogen, it completely inhibited the reaction. This result is not surprising, because glycerol is a tri-ol potentially capable of binding to the MIOX cofactor in the same manner as MI.

Discussion

Previous work had shown a large deuterium kinetic isotope effect of decay of G to

H, positioning H in the reaction sequence after abstraction of H• from C1. According to the four possible pathways summarized in Chapter 1 (Scheme1-6), species with

Fe2(II/III), Fe2(III/III) or Fe2(III/IV) cofactors would, in principle, have been viable candidates. The use of Mössbauer spectroscopy to define oxidation state of the diiron cluster in H therefore represents an important step toward its characterization. The isomer shifts of a high-spin Fe(II) site and a Fe(IV) site, which we would have expected to be in the high-spin configuration, due to the experimentally observed S =

½ ground state (11) and precedent from intermediate X from R2 (51), would be expected to differ by ~1 mm/s [δ ≈ 1.1-1.3 mm/s for high-spin Fe(II) and δ ≈ 0.2-0.3 mm/s for high-spin Fe(IV)]. Thus, the 120 K/zero field Mössbauer spectrum of the sample expected to contain the maximum fraction of H readily rules out the

Fe2(III/IV) possibility. Candidates with an Fe2(III/III) cluster, expected to have an S = 47

0 ground state and two sites with δ ≈ 0.35 – 0.60 mm/s, can be ruled out on the basis of the magnetic splitting in the spectrum of H and the observation of a spectral contribution from a site with a much larger δ.

In a recent paper by Morokuma (26), density function (DFT) and ONIOM quantum mechanical/molecular mechanical (QM/MM) calculations were applied to the MIOX reaction mechanism. In the proposed pathway (Scheme 2-2), the intermediate after

5 4

Scheme 2-2 A MIOX reaction pathway on the basis of DFT:MM study by Morokuma abstraction of H• form C1 by G was suggested to contain a hydroperoxo-Fe2(III/II) cluster and MI-1-one, resulting from a facile inner sphere electron transfer between the C1-centered radical and the hydroperoxo-Fe2(III/III) complex of the immediate product of the H•-abstraction step. The authors favored this structure for the identity of H (11), because conversion of this state via kinetically coupled nucleophilic attack of the peroxide on the C1 carbonyl and subsequent homolysis of the O-O-bond of the

Fe2(III/II) peroxyhemiketal complex had the highest calculated barrier. The observation in the chemical-quenched-flow experiments that DG, or some species that breaks down to DG upon the acidic denaturing quench, is produced as H forms,

provides strong argument against the Morokuma assignment. The MI-derived species in their assignment, MI-1-one is stable and should not break down to DG. Our results therefore suggest one of three possibilities: (1) the pathway deduced by Morokuma might be incorrect; (2) the relative energies or barriers to interconversion that they calculated might be incorrect; or (3) because they did not explicitly examine the barrier for dissociation of product, both their pathway and relative energies could be correct but product dissociation might just be much slower than even the rate-limiting chemical step. In scenario (3), H would be a product complex but distinct from the one that forms when DG is simply added to MIOX(II/III). We favor this possibility.

Chapter 3 Mechanism of the Reaction of the Fe2(II/II) Form of myo-Inositol Oxygenase [MIOX(II/II)] with O2 and H2O2 to Generate Active MIOX(II/III)

myo-Inositol oxygenase (MIOX) utilizes a non-heme di-iron cluster to activate its substrates, myo-inositol (cyclohexane-1,2,3,5/4,6-hexa-ol) and O2, for conversion to

D-glucuronate. Uniquely among all known non-heme di-iron oxygenases, MIOX uses the mixed-valent (II/III) oxidation state of its di-iron cluster, rather than the II/II state, as its functional cofactor. We previously reported that the reaction of the inactive fully reduced (II/II) form of MIOX [MIOX(II/II)] with limiting O2 activates the enzyme by converting it to MIOX(II/III) (9). This apparent one-electron oxidation of a diiron(II/II) cluster by O2 would also be unique, as all previously studied reactions of diiron(II/II) complexes (both enzymic and inorganic) with O2 have resulted in stepwise or concerted oxidation of both Fe sites of the cluster. In this work, we have determined the mechanism of this unusual activation reaction, showing that (1) O2 first affects a more conventional conversion of MIOX(II/II) to MIOX(III/III), with a stoichiometry of 2 MIOX(III/III)/O2; and (2) MIOX(III/III) then undergoes a slow compropotionation reaction with remaining MIOX(II/II), resulting in a limiting overall stoichiometry of 4 MIOX(II/II) + O2  4 MIOX(II/III). MIOX(II/III) accumulates to not more than  70%, apparently because it is (at least initially) in equilibrium with MIOX(II/II) and MIOX(III/III) and the reduction potential of the latter state is only  60 mV more positive than that of MIOX(II/III).

Introduction

Activation of dioxygen by non-heme di-iron proteins has been recognized for many years, and many members of this class have been extensively characterized (1-

5). In all well-characterized examples except one, the diiron(II/II) cluster reacts with

O2 to form a peroxo-Fe2(III/III) intermediate. This peroxide complex can either react directly with the substrate or undergo O-O-bond cleavage to produce a high-valent

[Fe2(IV/IV) or Fe2(III/IV)] complex which oxidizes the target. In all cases, a diiron(III/III) “product” state of the cluster is generated as part of each O2-activation event. For subsequent events to occur, the cofactor must be returned to the “reactant” diiron(II/II) state, with electrons provided ultimately by NAD(P)H. This cycling of the cofactor and oxidation of a nicotinamide cosubstrate ensure that at most two electrons can be extracted from the substrate.

Myo-inositol oxygenase (MIOX) uses its non-heme di-iron cluster to catalyze the four-electron oxidation of myo-inositol (cyclohexane-1,2,3,5/4,6-hexa-ol; MI) by

1 equiv of O2 (16), producing D-glucuronate. No external reducing equivalents are required. These two characteristics make the MIOX reaction distinct from those effected by other family members. At the outset of my Ph.D. work, we discovered and reported that the active form of MIOX is MIOX(II/III), is actually the state to activate O2 and competent for D-glucuronate production (11). This implication of use of the diiron(II/III) cofactor is consistent with several of the previously reported complex kinetic characteristics of MIOX such as the activity enhancement by L-Cys

(20). We have shown the evidence that MIOX(II/II) is not the catalytically active

enzyme form (11), which would imply that its reaction with O2 to generate the mixed- valent state is an activation step. An interesting and fundamental question is how the enzyme is activated from its fully reduced form MIOX(II/II). Is it a direct one-

electron oxidation step from the

reduced form or more complex

Scheme 3-1 One possible MIOX(II/II) process associated? On the basis of

activation mechanism in which H2O2 is formed the reaction stoichiometry (Figure 3- in the first step. 1) when MIOX(II/II) reacts with various amount of O2, a mechanism (Scheme 3-1) for the activation of MIOX(II/II) was proposed. In this chapter we have demonstrated the process in which

MIOX(II/III) is produced from MIOX(II/II) reaction with dioxygen. UV-vis absorption, EPR, and Mössbauer spectroscopies provide extensive evidence regarding how this process occurs. Moreover, the kinetics in this process has also been investigated.

Materials and Methods

Preparation of MIOX (III/III).

Preparation of fully oxidized state of the enzyme (III/III) was carried out by substoichiometric addition of Fe(II)aq [the ammonium sulfate salt for natural- abundance Fe(II) and the sulfate salt prepared by dissolution of Fe(0) in H2SO4 for

57Fe(II)] to 30 μM ambient solution of the iron-free MIOX protein and concentrating to appropriate concentration [giving a greater than 95% yield of MIOX(III/III)].

O2 Quantification. 53

Quantity of O2 (g) were calculated according to the ideal gas law. The solubility of

O2 in MIOX buffer [50mM Bis-tris-acetate (pH 6.0) and 10% (w/w) glycerol] for calculation of the O2 concentration in reactant solution is described previously (11).

Stoichiometry of O2 Reaction with MIOX (II/II).

57 An O2-free solution containing 3 mM MIOX and 5.4 mM Fe(II) was rapidly

o o mixed at 5 C with 0.5 eq. volume of O2-free, air-saturated (5 C), O2-saturated (50 o o o C), O2-saturated (23 C), and O2-saturated (5 C) buffer respectively (giving 0, 0.09,

0.19, 0.295 and 0.454 mM O2). The reaction solution was sealed inside the reaction hose, incubated at 5 oC for 1 hour, taken into the anoxic chamber, added with 100 mM MI, transferred to a Mössbauer sample cup, and frozen for Mössbauer analysis.

Similar experiment with lower concentration of enzyme was carried out to cover the O2/MIOX(II/II) ratio in the range from 0 to 1 and analyzed with EPR spectroscopy. The integrated intensity of the axial g < 2 EPR signal at a subsaturating microwave power was compared to the intensity of the spectrum of a copper(II) perchlorate standard, as previously described (52, 53) for determination of concentration of the produced MIOX(II/III). The spectrum of buffer was acquired so that the contribution of the EPR instrument cavity could be subtracted. An O2-free solution containing 3 mM MIOX and 6 mM Fe(II) was rapidly mixed at 5 oC with

o o either 0.5 equiv volume of O2-free, air-saturated (5 C), O2-saturated (23 C) and O2-

o o o saturated (5 C) or 1 equiv volume of O2-saturated (23 C) and O2-saturated (5 C) or

o 2 equiv volume of O2-saturated (5 C) buffer respectively (giving 0.03, 0.14, 0.37,

0.52, 0.51, 0.74 and 0.98 mM O2). The reaction solution was sealed inside the

reaction hose, incubated at 5 oC for 1 hour, taken into the anoxic chamber, transferred to an EPR tube, and frozen for EPR analysis.

The simulation of different oxidation state percentage in the reaction with different

O2/MIOX (II/II) ratio (colored solid line overlaid with the experiment data in Figure

3-1 B and Figure 3-2) was performed using KinTekSim (KinTek Corp., Austin, TX).

The mechanism and parameters are presented in Scheme 3-1.

57 Stoichiometry of H2O2 Reaction with Fe MIOX (II/II).

57 An O2-free solution containing 3 mM MIOX and 4.8 mM Fe(II) was rapidly

o mixed at 5 C with equal volume of 0.75, 1.5, 2.25, 3 and 6 mM H2O2 solution respectively (giving 0, 0.25, 0.5, 0.75, 1 and 2 equiv of H2O2). The reaction solution was incubated in a reaction tube at 5 oC for 1 hour in the anoxic chamber, added with

100 mM MI, transferred to a Mössbauer sample cup, and frozen. The anaerobic control sample was made by hand-mixing of equal volume of MIOX (II/II) with O2- free buffer followed by 100 mM MI addition. The similar simulation procedure as described above in O2 reaction was used to compare with experimental data and imply mechanism (colored solid lines overlaid with experimental data in Figure 3-3 B).

Kinetic and Spectroscopic Experiments of Reaction of MIOX (II/II) with O2 or H2O2

The stopped-flow (SF) apparatus, the freeze-quench (FQ) apparatus and procedures have been described previously (50). In a SF experiment to define the kinetics of reaction of MIOX (II/III) with O2 or H2O2, MIOX(II/II) was prepared by addition of 2

equiv of Fe(II) to the iron-free MIOX. This solution was mixed at 5 oC with an equal volume of solution of O2 or H2O2. Absorbance-versus-time traces from the reactions of MIOX(II/II) with O2 were analyzed by nonlinear regression according to eq 1, in which k1 and k2 are apparent first-order rate constants, ΔA1 and ΔA2 are amplitudes for the exponential phases, and A0 is the absorbance at time zero. The assumption of a pseudo-first-order excess of O2 or H2O2 inherent in these equations is met by the experimental conditions.

In a FQ Mössbauer experiment, MIOX(II/II) was prepared by addition of 1.8 equiv of 57Fe(II) to iron-free MIOX. An aliquot was frozen in a Mössbauer cup for determination of the concentration of MIOX(II/III) in the reactant solution from the contamination of O2 in the anoxic chamber. The MIOX(II/II) solution was mixed at 5 o o C with 2 equiv volume of O2-saturated (5 C) or equal volume of excess H2O2

(giving 0.6 mM MIOX and 0.95 mM O2 in excess-O2 samples or 0.9 mM MIOX and

10 mM H2O2 in excess-H2O2 samples) and the reaction solution was freeze-quenched after desired times. The total reaction time was calculated as the sum of the known time for transit through the aging hose and the “quench time” (the time required for cooling to a temperature at which no further reaction occurs) of 0.015 s. Following preparation of one of the samples, the reaction solution remaining in the hose was sealed inside the hose, incubated at 5 oC for 10 min, taken back into the anoxic chamber, transferred to a Mössbauer cup, and frozen for Mössbauer analysis. To obtain the kinetics of the MIOX(II/II) reactant from the time-dependent spectra, the

absorbance at 370 nm was used to represent the formation of MIOX(III/III). In FQ

Mössbauer spectra, a constant contribution of MIOX(II/III) in the MIOX(II/II) reactant was removed from each freeze-quenched sample. In each sample, contributions of MIOX(II/II), and MIOX(III/III) were obtained from simulation using

WMOSS (WEB Research, Edina, MN).

Reaction of MIOX(II/III) with Limiting O2

o An O2-free solution of 0.96 mM MIOX (II/II) was rapidly mixed at 5 C with equal volume of air-saturated (5 oC) buffer giving a final concentration of 0.48 mM MIOX

(II/II) and 0.11 mM O2. The kinetics of MIOX (II/II) activation by limiting O2 was then investigated by absorption and EPR spectroscopies. In FQ EPR experiment, an aliquot of reactant MIOX(II/II) solution was frozen in an EPR tube for determination of the concentration of MIOX(II/III) in the reactant solution (blue spectrum in Figure

3-10 B). The reaction shorter than 10 min was quenched as described in above section.

Longer reaction solution was sealed inside the reaction hose, incubated at 5 oC for desired time, taken into the anoxic chamber, transferred to an EPR tube, and frozen for EPR analysis. The concentrations of MIOX(II/III) in each sample was determined as described above.

The reaction of MIOX(II/II) with limiting O2 was also analyzed with FQ

Mössbauer experiment. A solution of 3.3 mM O2-free MIOX(II/II) was rapidly mixed

o o at 5 C with 1 equiv. volume of O2-saturated buffer (23 C) containing 0.8 mM O2, and the reaction solution was freeze-quenched after desired time (Figure 3-9). An

aliquot of MIOX(II/II) was mixed with 1 equiv volumes of O2-free buffer and frozen

in an Mössbauer cup for determining the contribution of MIOX(II/III) in the reactant

MIOX(II/II). The concentrations and spectroscopic conditions are given in the

corresponding figure legends.

Comproportionation Reaction of MIOX(II/II) with MIOX(III/III)

In a typical comproportionation reaction, a solution of 2 mM O2-free MIOX (III/III)

prepared in a procedure described previously was mixed with equal equivalent of

MIOX (II/II) and incubated for 4 hours to get a MIOX (II/III) enriched solution.

Details of the reaction are given in the figure legends.

An isotope swap experiment was performed to demonstrate comproportionation

57 and disproportionation reactions. Equal equivalent of O2-free Fe MIOX (II/II) and

56Fe MIOX (III/III) [preparations of MIOX (II/II) and MIOX(III/III) were described

previously(9)] were mixed at 5 oC, quenched with excess MI after desired times to

make Mössbauer samples. Details of the reaction are given in the figure legends.

Attempt to Directly Trap H2O2 as a Product of MIOX(II/II) Reaction with O2

In a FQ apparatus which have been described previously (49) one volume of 0.48

o mM MIOX(II/II) was rapidly mixed with 2 volume of O2-saturated buffer (5 C) to give a final concentration of 0.16 mM MIOX(II/II) and 0.95 mM O2. After 100 s the reaction mixture was pushed into a centrifuge tube containing horse radish peroxidase

(HRP) colorimetric assay solution. The assay to determine H2O2 concentration has

been described previously (54). A control sample was prepared in the same procedure but with addition of a known amount of H2O2 into assay solution.

EPR and Mössbauer Spectroscopy

The spectrometers have been described previously (50). Specific conditions are

given in the appropriate figure legends. Simulation of Mössbauer spectra was carried

out using WMOSS.

Results

Stoichiometry of O2 Reaction with MIOX(II/II)

It was demonstrated that MIOX(II/III)•MI yields better resolved Mössbauer

features than MIOX(II/III) but does not change the distribution of different oxidation

states of diiron cluster (9). Thus, to quantitatively determine the contribution of

MIOX(II/III), MI was added at the end of the reaction. In the reactant solution

containing MIOX(II/II) state, there is a 13% contribution of MIOX(II/III) due to the

contamination of O2 in the anoxic chamber (top spectrum in Figure 3-1 A). In the

experiment of MIOX(II/II) with O2 under different O2/MIOX(II/II) ratio, when the

equilibrium is reached, the yield of MIOX(III/III)•MI, MIOX(II/III)•MI and

MIOX(II/II)(•MI)are different. The formation of MIOX(III/III)•MI (green line in

Figure 3-1 A) increases intensity with increasing O2/MIOX(II/II) ratio. By contrast,

MIOX(II/II)(•MI) (quadrupole doublet in top spectrum of Figure 3-1A), the majority

state in reactant solution, decreases its intensity when O2/MIOX(II/II) ratio becomes 59

A B

Figure 3-1 Mössbauer analysis of MIOX(II/II) activation by various amount of O2. A. Mössbauer spectra of MIOX samples recorded at 4.2-K in a 53-mT magnetic field

applied parallel to the γ-beam. These samples were prepared as followed: an O2-free solution of 3 mM MIOX and 5.4 mM 57Fe(II) was rapidly mixed at 5 oC with a buffer

containing different concentration of O2 to give final (O2/MIOX ratios of 0.032, 0.085, 0.14, 0.20 and 0.28), sealed in a reaction hose at 5 oC for 1 hour and added with 100 mM MI at the end in the anoxic chamber. The blue and green solid lines overlaid with the data are the contributions of MIOX(II/III)⦁MI and MIOX(III/III)⦁MI respectively. The color-coded arrows indicate the features of corresponding species. B. The fraction of different oxidation states of diiron cluster has been plotted with colored dots as the

function of O2/MIOX ratio. Solid lines corresponding to different oxidation states are the simulation according to the model mechanism showed in Scheme 3-1. The error bars are the uncertainties of the Mössbauer analysis. higher. MIOX(II/III)•MI (blue line in Figure 3-1A) yield increases when

O2/MIOX(II/II) ratio changes from 0 to 0.2 and starts to decease (or does not change considering uncertainty of Mössbauer analysis) in the 0.28 equiv of O2 sample. Using the reference spectra of MIOX(II/III)⦁MI and MIOX(III/III)⦁MI generated previously

(9), the contributions of different oxidation states with substrate bound can be

obtained and plotted in Figure 3-1B versus O2/MIOX(II/II) ratio. The simulation of the yield of different states on the basis of the proposed mechanism (see below for description of the mechanism and parameters in Scheme 3-1) is also plotted as colored solid lines in Figure 3-1B. The experimental result is consistent with the simulation and two things caught interest of us immediately. First, similar to the yield of the other methods, the yield of MIOX(II/III) is 60% -70% and in this experiment when the highest point is reached, the concentrations of MIOX(II/II) and

MIOX(III/III) are equivalent (20% or 15% of each). An yield of 60% - 70%

MIOX(II/III) is by far the highest we have obtained regardless of methods [O2 diffusion from MIOX(II/II), reduction from MIOX(III/III) using reductant and comproportionation (see below)] applied. So it could indicate that equilibrium is reached and limited by the reduction potential of different states. Second, the optimal yield of MIOX(II/III) is obtained when O2/MIOX ratio is close to 0.25 although higher ratio points were missed in this set of data due to the protein concentration requirement for Mössbauer sample and limited O2 solubility. These observations can be interpreted with a proposed activation mechanism initiated from MIOX(II/II)

(Scheme 3-1). In this model, MIOX(II/II) first reacts with 1 molecular of O2 to form one MIOX(III/III) and an H2O2. Subsequently a second molecule of MIOX(II/II) can react with the formed H2O2 to generate a second MIOX(III/III). At last, two molecules of formed MIOX(III/III) as intermediate comproportionate with two molecules of MIOX(II/II) to give four molecules of MIOX(II/III). This model to great extent interprets the outcome of stoichiometry experiment when O2/MIOX is no

larger than 0.3. In the net reaction, 1 equiv of O2 generates 4 equiv of MIOX(II/III) thus O2/MIOX ratio value of 0.25 is the optimal number to yield the maximal

MIOX(II/III). The model depict a relationship between the yield of MIOX(II/III) and

O2/MIOX ratio. When O2/MIOX is less than 0.25, MIOX(II/II) has higher amount than MIOX(III/III) and MIOX(II/III) keeps increasing till the optimal value of 0.25 is reached. After this point, more MIOX(III/III) is converted from MIOX(II/II) and disfavors the formation of MIOX(II/III) by disturbing the equilibrium. When

O2/MIOX equals or becomes higher than 0.5, all MIOX(II/II) oxidizes to

MIOX(III/III) and no MIOX(II/III) is generated under this condition.

To have a full coverage of O2/MIOX ratio from 0 to 1, less-protein-required EPR samples were prepared and analyzed to determine the concentration of formed

MIOX(II/III) (Figure 3-2). When the ratio is less than 0.25, experimentally formed

MIOX(II/III) is consistent with the simulation based on the model (Scheme 3-1).

However, when O2/MIOX ratio is more than 0.25, the formation of MIOX(II/III) does

Figure 3-2 The fraction of MIOX(II/III) in total diiron cluster determined by EPR analysis (closed circles) was

plotted as the function of O2/MIOX ratio. Each sample

was prepared by the rapid mixing of O2-free MIOX(II/II) with buffer containing different

concentration of O2 at different mixing ratio and subsequent incubation at 5 oC for 1 hour. The only EPR sensitive species MIOX(II/III) was quantified with cooper standard and compared with the simulation according to model mechanism (Scheme 3-1).

not drop significantly as expected. The yield of MIOX (II/III) is ~ 60% when

O2/MIOX ratio is close to 0.5 under which condition all MIOX(II/II) should be oxidized to MIOX(III/III) and no MIOX(II/III) would be formed according to the model in Scheme 3-1.

Stoichiometry of H2O2 Reaction with MIOX(II/II)

Similar experiment with H2O2 as the reactant has been analyzed with Mössbauer spectroscopy. According to the model in Scheme 3-1, if the activation is initiated with

H2O2, the stoichiometry of MIOX(II/III) formation with increasing concentration of

H2O2 shows distinction from O2 activation; optimal yield (~ 70%) is the outcome when H2O2/MIOX ratio approaches 0.5 (colored lines in Figure 3-3B). Mössbauer analysis, however, indicates an inconsistent outcome than model but similar as O2 activation experiment result. Before the point where optimal MIOX(II/III)•MI is formed, experimental data falls on the simulation curve perfectly. By contrast, higher

H2O2/MIOX generates more MIOX(II/III)•MI than expected, the formation of

MIOX(II/II)(•MI) and MOX(III/III)•MI is also inconsistent with the simulation derived from model mechanism.

Figure 3-3 Mössbauer analysis of MIOX(II/II) activation by various amount of H2O2. A. Mössbauer spectra of MIOX samples recorded at 4.2-K in a 53-mT magnetic field

applied parallel to the γ-beam. These samples were prepared as followed: an O2-free solution of 3 mM MIOX and 4.8 mM 57Fe(II) was rapidly mixed with a buffer

containing different concentration of H2O2 to give final H2O2/MIOX ratios of 0, 0.25, 0.50, 0.75, 1 and 2, sealed in a reaction hose at 5 oC for 1 hour and added with 100 mM MI at the end in the anoxic chamber. The blue and green solid lines overlaid with the data are the contributions of MIOX(II/III)⦁MI and MIOX(III/III)⦁MI respectively. B. The fraction of different oxidation states of diiron

clusters has been plotted with colored dots as the function of H2O2/MIOX ratio. Solid lines corresponding to different oxidation states are the simulations according

to the model mechanism initiated with H2O2 showed in Scheme 3-1.

Kinetics of the Reaction of the MIOX(II/II) with Excess O2 by Stopped-Flow

Absorption and Freeze-Quench Mössbauer Spectroscopies.

Stopped-flow absorption and freeze-quench Mössbauer experiments were used to define the kinetics of the reaction of MIOX(II/II) with O2. Intense bands at ~ 370 nm 64

and ~ 560 nm associated with MIOX(III/III) develops rapidly upon mixing of

MIOX(II/II) with O2 a and then reaches plateau (Figure 3-4A). Analysis of A370- versus-time traces for the reaction (Figure 3-4B) according to eq 1 reveals that the

A B

Figure 3-4 Kinetics of the reaction of the MIOX(II/II) with excess O2. A. A sample of the MIOX(II/II) (0.3 mM) solution was mixed at 5 oC with an equal volume of buffer containing 0.95 mM O2. The concentration after mixing were 0.15 mM MIOX(II/II), o 0.48 mM O2. B. A same solution of MIOX(II/II) was mixed at 5 C with equal colume of buffer containing various concentration of O2. The final O2 concentrations are 0.24 mM (red), 0.48 mM (blue) and 0.71 mM (green). The solid lines are fits according to eq 1. The inset shows the fastest apparent first-order rate constants for the formation phase vs 3 -1 O2 concentration, which gives a second-order rate constant (slope) of 1.8 × 10 ± 20 M s-1. formation phase has a sensitive first-order dependence on O2 concentration (inset of

Figure 3-4B). The slope of the plot of the apparent first-order rate constant for the fastest formation phase (kobs) versus O2 concentration gives a second-order rate

3 -1 -1 constant of 1.8 × 10 ± 20 M s for the reaction of the MIOX(II/II) with O2.

The formation of MIOX(III/III) was directly demonstrated by freeze-quench

Mössbauer experiments (Figure 3-5). In a reaction of MIOX(II/II) with excess O2, the

4.2-K/53-mT spectra of samples that were rapidly frozen after desired times, exhibit

increasing contribution from MIOX(III/III) (green quadrupole doublet in Figure 3-5A with parameters δ of 0.48 mm/s and ΔEQ of 1.01 mm/s) in 0.46 s (47 ± 3%), 2 s (62 ±

3%) and 6 s (80 ± 3%) samples, and has slight decrease in 20 s sample (64 ± 3%). In

Figure 3-5 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of

MIOX(II/II) with excess O2. The anaerobic control sample (top spectrum) contained 2 mM MIOX, 3.6 mM 57Fe(II). The reaction of this reactant MIOX(II/II) with 2 equiv o volume of O2-saturated buffer (5 C) gave a final concentration of 0.6 mM MIOX(II/II)

and 0.95 mM O2, and was rapidly freeze-quenched after desired times. A. The solid lines overlaid with the data are the sum of three contributions from MIOX(II/II), MIOX(II/III) and MIOX(III/III) using the reference spectra. The solid lines above the data are the contributions of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B. The kinetics of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). The solid lines are fits of the single exponential to the data. First-order rate constants of 2.0 ± 0.4 s-1 and 2.4 ± 1.0 s-1 were extracted from the fits to the MIOX(II/II) decay and the MIOX(III/III) formation, respectively. The error bars are the uncertainties of the Mössbauer analysis.

the starting MIOX(II/II) sample, a less than 4% contribution is from the MIOX(II/III) due to the contamination of O2 in the anoxic chamber. MIOX(II/III) amount (blue line in Figure 3-5A) increases in a slow and weak fashion. The intensity of

MIOX(II/II) (red quadrupole doublet in Figure 3-5A with parameters δ of 1.31 mm/s and ΔEQ of 3.05 mm/s) decreases rapidly but 14 ± 4% unreacted MIOX(II/II) remains in the 20 s sample. It could be attributed to a small fraction of MIOX(II/II) that does not react rapidly. The broad quadrupole doublet of MIOX(II/II) (with the width, Γ, of 0.7 mm/s) also indicates its heterogeneity nature. The kinetic data of the reaction (Fig 3-5B) were analyzed as single exponential fits. First-order rate constants of 2.0 ± 0.4 s-1 and 2.4 ± 1.0 s-1 were obtained from the fits and a second- order rate constant was readily determined as a value of 2.5 × 103 ± 1.0 × 103 M-1 s-

1 which is consistent with the data from the SF absorption spectroscopy (1.8 × 103 ±

20 M-1 s-1).

Kinetics of the Reaction of the MIOX(II/II) with H2O2 by Stopped-Flow Absorption and Freeze-Quench Mössbauer Spectroscopies.

Similarly as the reaction of MIOX(II/II) with excess O2, in the reaction of

MIOX(II/II) with excess H2O2, absorption bands at ~ 370 nm and ~ 560 nm associated with MIOX(III/III) developed rapidly upon mixing of MIOX(II/II) with

H2O2 and then reached a plateau (Figure 3-6A). Analysis of A370-versus-time traces for the reaction (Figure 3-6B) according to eq 1 reveals that the formation phase has a sensitive first-order dependence on H2O2 concentration (inset of Figure 3-6B). The

slope of the plot of the apparent first-order rate constant for the fastest formation

3 phase (kobs) versus H2O2 concentration gives a second-order rate constant of 2.1 × 10

-1 -1 3 ± 20 M s very close to the value of the reaction of MIOX(II/II) with O2 (1.8 × 10

A B

Figure 3-6 Kinetics of the reaction of the MIOX(II/II) with excess H2O2. A sample of the MIOX(II/II) (0.3 mM) solution was mixed at 5 oC with an equal volume of buffer

containing H2O2 (10 mM for panel A and 10 mM, 30 mM and 90 mM for panel B) to

give a final concentration of 0.15 mM MIOX(II/II), 5 mM H2O2 in panel A and 5 mM (red), 15 mM (blue) and 30 mM (green) in panel B. B. The solid lines are fits according to eq 1. The inset shows the fasted apparent first-order rate constants for the formation

phase vs H2O2 concentration, which gives a second-order rate constant (slope) of 2.1 × 103 ± 20 M-1 s-1.

± 20 M-1 s-1).

The oxidation of MIOX(II/II) by H2O2 is also directly demonstrated by

Mössbauer spectroscopy. The MIOX(II/II) (red line in Figure 3-7A), the majority species in the starting solution (> 96%), rapidly decayed after mixing with excess

H2O2. By the contrast, the intensity of MIOX(III/III) (green line in Figure 3-7A) increased and reach the maximum in 0.3 s. Surprisingly, MIOX(II/III) (blue line in

Figure 3-7A) formed rapidly (25 ± 6% in 0.02 s) but retained a constant contribution in the proceeding reaction. The kinetics of MIOX(II/II) decay and MIOX(III/III)

A B

Figure 3-7 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of

MIOX(II/II) with excess H2O2. The anaerobic control sample (top spectrum) contained 2 mM MIOX, 3.6 mM 57Fe(II). The reaction of this reactant MIOX(II/II) was rapidly mixed o at 5 C with equal volume of buffer containing 20 mM H2O2 gave a final concentration of

0.9 mM MIOX(II/II) and 10 mM H2O2, and was rapidly freeze-quenched after desired times. A. The solid lines overlaid with the data are the sum of three contributions from MIOX(II/II), MIOX(II/III) and MIOX(III/III) using the reference spectra. The solid lines above the data are the contributions of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B. The kinetics of MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). The solid lines are fits of single exponential equations to the data and first-order rate constants of 44 ± 21 s-1 and 22 ± 8 s-1 were extracted for the decay of MIOX(II/II) and formation of MIOX(III/III), respectively. C. Overlay of Mössbauer spectra of the diiron(III) clusters in 0.02 s (green) and 10 min (blue) freeze-quenched samples. The spectrum of the 0.02 s sample was prepared by removing the 25% MIOX(II/III) and 39% MIOX(II/II) contributions from the raw spectrum of the 0.02 s sample, and the spectrum of the 10 min sample was prepared by removing 25% MIOX(II/III). The solid lines are theoretical spectra simulated with parameters of the Fe species reported in the text.

formation (red and green traces in Figure 3-7B respectively) were analyzed as single exponential equations and obtained first-order rate constants of 44 ± 21 s-1 and 22 ± 8

-1 s , respectively. Given that 10 mM H2O2 was mixed with MIOX(II/II), a second- order rate constant of 2.2 × 103 ± 800 M-1 s-1 for formation of MIOX(III/III) is consistent with the value of 2.1 × 103 ± 20 M-1 s-1 from SF absorption spectroscopy.

We emphasize that simple mechanism for MIOX(II/II) oxidation by either O2 or

H2O2 [one-step, irreversible oxidation to MIOX(III/III)] does not adequately account for the data. In both O2 and H2O2 reaction, there is a second slow formation phase that is required further characterization. In both O2 and H2O2 cases, MIOX(II/III) was formed in a small fraction and did not decay. It is not yet clear whether this kinetics complexity reflects multistep oxidation of MIOX(II/II) or any intermediate formation during the reactions. The small gap between the decay of MIOX(II/II) and the formation of MIOX(III/III) (Figure 3-7B) in the reaction of MIOX(II/II) with excess

H2O2 may imply an intermediate formed in the reaction. The precedents of possible intermediates are peroxo diiton(III/III) complex in sMMO (55) or diiron (III/III) intermediate in ToMOH (56). Three reasons could be responsible for scarce accumulation of distinguished intermediate in FQ Mössbauer spectroscopy: (1) if the formation of the intermediate is not kinetically favorable and be able to accumulate to certain extent under experiment condition, (2) if the diiron(III/III) intermediate is not spectroscopically discernible from the diiron(III/III) product and, (3) if the difference of two diiron(III/III) center is too small to be identified. Figure 3-7C shows a small shift of quadrupole doublets of diiron(III/III) species in 0.02 s (green) and 10

min (blue) samples with the parameters δ of 0.46 mm/s, ΔEQ of 1.04 mm/s in 0.02 s sample and δ of 0.49 mm/s and ΔEQ of 0.98 mm/s in 10 min sample. This is an analogy of the small shift of diiron(III/III) intermediate from the diiron(III/III) product during the reaction with dioxygen of the reduced hydroxylase component of

ToMOH (56), although the difference of ΔEQ value (0.06 mm/s) in the MIOX case is not as pronounce as the difference in ToMOH (0.18 mm/s).

Failure to Detect H2O2 in the Reaction of MIOX(II/II) with Excess O2.

According to the model shown in

Scheme 3-1, H2O2 is generated in the reaction of MIOX(II/II) with O2. In a reaction of MIOX(II/II) with excess O2, given that the MIOX(II/II) oxidation by O2

3 -1 -1 (1.8 × 10 M s ) is comparable with Figure 3-8 H2O2 concentration

3 dependence of the absorbance at 490 MIOX(II/II) oxidation by H2O2 (2.1 × 10 nm of the quinone imine dye, -1 -1 M s ) and much faster than the following generated by the HRP assay. comproportionation (shown later), H2O2 should accumulate to 0.14 mM after 100 s and be readily detected by HRP colorimetric assay (54). HRP colorimetric assay can detect as low as ~ 30 µM H2O2 (Figure 3-8). However, no absorbance was detected by this sensitive assay in the reaction mixture, which suggests that no more than 30

µM H2O2 was generated in the reaction. Thus, H2O2 should not be a product of the reaction of MIOX(II/II) with O2 and model in Scheme 3-1 would not precisely

describe the activation of MIOX(II/II) by O2. Alternative mechanism is worthy of investigation.

Reaction of MIOX(II/II) with Limiting O2 Implied MIOX(III/III) as an Intermediate

Approaching MIOX(II/III) Active Form

The mixed-valent diiron(II/III) state of MIOX was discovered by accident in which the color of fully reduced MIOX(II/II) changed from almost colorless to pink when it was placed in the anoxic chamber and assumed as the catalytically active form of the enzyme. To date, the catalytic mechanism of MIOX has been extensively studied (57) thus the conversion of MIOX(II/II) to MIOX(II/III) is considered as an activation process and was applied to prepare MIOX(II/III) (11). In this study, freeze-quench

Mössbauer spectroscopy demonstrated the activation process by illustrating the change of three oxidation states of diiron clusters. Anaerobic control sample was prepared by direct addition of 1.9 equiv of 57Fe(II) to apo MIOX. The 4.2-K/53-mT spectrum of the anaerobic control sample shows that majority of the sample is

MIOX(II/II) (90%, red solid line in Figure 3-9A) and 10% MIOX(II/III) (blue solid line in Figure 3-9A) is from the reaction of MIOX(II/II) with contaminated O2 in the anoxic chamber. A quadrupole doublet with δ = 0.49 mm/s and ΔEQ = 1.01 mm/s

(green solid line in Figure 3-9A) developed in 0.35 s sample (22 ± 3%) is attributed to the previously characterized MIOX(III/III) (9). The MIOX(III/III) actually accumulated to the maximum after 4 s (44 ± 3%) then decayed to 23 ± 3% (in 166 s) and 16 ± 3% (in one hour). As the consequence, MIOX(II/II) (a red quadrupole

Figure 3-9 A: 4.2-K/ 53-mT Mössbauer spectra of MIOX samples from the reaction of

MIOX(II/II) with limiting O2. A reactant MIOX(II/II) was prepared by addition of 6.6 57 mM Fe(II) to 3.5 mM O2-free apo MIOX and frozen to make an anaerobic control sample. The reaction of this reactant MIOX(II/II) with equal volume of buffer containing

0.8 mM O2 gave a final concentration of 1.66 mM MIOX(II/II) and 0.4 mM O2, and was rapidly freeze-quenched after desired times. The solid lines overlaid with the data are the sum of theoretical reference spectra of different diiron forms with different contributions reported in the text, and the color-coded solid lines above the data are the contributions of the MIOX(II/II) (red), MIOX(II/III) (blue) and MIOX(III/III) (green). B: Kinetics of the different diiron clusters [MIOX(II/II) in red, MIOX(II/III) in blue and MIOX(III/III) in green] in the reaction. The solid lines are simulations of the kinetic data according to Scheme 3-2. Error bars are the uncertainties of Mössbauer analysis. doublet with δ = 1.31 mm/s and ΔEQ = 3.24 mm/s in Figure 3-9A) decayed from 90 ±

3% in starting material sample to 63 ± 3% (in 0.35 s), 35 ± 3% (in 4 s), 33 ± 3% (in

166 s) and 16 ± 3% (in 1 hour). In addition, absorption of MIOX(II/III) with broad features in 4.2-K spectra (blue solid line in Figure 3-9A) increased with increasing time (15 ± 5% in 0.35 s, 21 ± 5% in 4 s, 44 ± 5% in 166 s, and 68 ± 5% in 1 hour).

After 1 hour, the reaction approached equilibrium and later reaction time sample

(after 2 hour) had slight variation in contributions of different oxidation states (data not shown). These data can be rationalized by a model in which MIOX(III/III) is converted fast from MIOX(II/II) as an intermediate and then slowly comproportionates with remaining MIOX(II/II) to generate MIOX(II/III). This model suggests that MIOX(II/III) is not produced directly from MIOX(II/II) via one-electron oxidation but by the comproportionation of MIOX(II/II) with an intermediate

MIOX(III/III). In addition, one molecular of O2 can react with excess MIOX(II/II) to generate 4 molecules of MIOX(II/III) at the theoretical circumstances. However, due to the thermodynamic limit between the standard reduction potential for the III/III →

II/III step and the II/III→ II/II step (~ 60 mV), maximum yield of MIOX(II/III) is not higher than 65-70% initiating with either MIOX(II/II) by oxidation or MIOX(III/III) by L-cysteine (or the alternative reductant) reduction. Using the mechanism and kinetic parameters in Scheme 3-2 (see discussion), the simulation agrees fairly well with the kinetics of experimental diiron fractions (Figure 3-9B), even though the rate constants have not been well determined.

The process of MIOX(II/III) production from MIOX(II/II) was also assessed by SF absorption spectroscopy in which MIOX(II/II) was mixed with limiting O2. Time- dependent absorption spectra acquired after the mixing (Figure 3-10A) show transient features at ~ 600 nm and ~370 nm associated with the formation and decay of

MIOX(III/III) (11). The decrease of absorbance at 600 nm is much slower compared with its increase and is coupled with a positive feature at 495 nm which is known as

characteristic of substrate-free MIOX(II/III) (11). To precisely trap the formation delay of MIOX(II/III), a FQ EPR experiment was performed under the same condition as SF experiment (Figure 3-10B). MIOX(II/III) is the only detectable state due to its S = ½ property. It is clear that MIOX(II/III) features at g = 1.95, 1.66 and

A B C

Figure 3-10 Kinetics of reaction of MIOX(II/II) with limiting O2. In both SF and FQ experiment, a solution of 0.96 mM MIOX(II/II) was rapidly mixed at 5 oC with equal volume of air-saturated buffer (5 oC) to give a final concentration of 0.48 mM MIOX(II/II) and 0.11 mM O2. A. Reaction of MIOX(II/II) with limiting O2 monitored by SF absorption spectroscopy. The arrows indicate the changes of absorbance at ~ 600 nm and ~ 495 nm. B. X-Band EPR spectra of samples freeze-quenched at various reaction times during the limiting O2 reaction. The spectra were acquired at 10 K (nominal temperature on an Oxford cryostate controller) with a microwave frequency of 9.45 GHz, a microwave power of 20 mW, a modulation frequency of 100 kHz, a modulation amplitude of 10 G, a time constant of 327 ms, and a scan time of 167 s. The spectrum of a sample containing buffer alone was subtracted from all spectra to remove the feature intrinsic to the EPR cavity at 3380 G (g = 2.00). C. Kinetics of absorbance at 600 nm (dots, left y-axis) and of MIOX(II/III) (triangles, right y-axis) in the reaction. Calculation of the MIOX(II/III) concentrations from the intensities of the EPR features was described in Materials and Methods. The solid lines are simulations of the kinetic data according to Scheme 3-2.

1.66 shows significant intensity increase only after 100 s and is slowly produced more till a steady equilibrium approaches. It is consistent with optical absorption and

FQ Mössbauer spectra and indicates that MIO(II/III) was generated later than

MIOX(III/III). The simulation according to the kinetics shown in Scheme 3-2 is relatively consistent with the experimental data (Figure 3-10C). Based on the fact that

MIOX(III/III) is formed faster and sooner than the formation of MIOX(II/III), we can tentatively concludes that activation step for generating mixed-valent state is not a direct one electron oxidation from MIOX(II/II).

Direct Evidence of Comproportionation and Discomproportionation.

Simply mixing equal equiv of MIOX(II/II) with MIOX(III/III) and adding MI in an hour generate a sample having more complex Mössbauer features than starting materials (Figure 3-11A). It was a slow process up to 1 hour. The spectrum reveals that this sample has 65 ± 5% MIOX(II/III)•MI and equal amount of MIOX(II/II)(•MI) and MIOX(III/III)•MI with 17 ± 3% contribution respectively. The MIOX(II/III) prepared by comproportionation method shows identical spectroscopic features

(UV/Vis, EPR and Mössbauer) with those from the MIOX(II/III) prepared with other methods (oxidation or reduction). Kinetically, this MIOX(II/III) has full activity (kcat

= 0.7 s-1) and the MIOX(II/III)•MI complex is fully competent to initiate the reaction.

The comproportionation method yields similar amount of MIOX(II/III) as oxidation and reduction methods, which could be due to the thermodynamic upper limit and also imply a reversible discomproportionation step.

An isotope swap experiment strongly supports the validity of this step. Equally mixing 57Fe MIOX(II/II) with 56Fe MIOX(III/III) for desired time and adding MI

shows increasing MIOX(III/III)•MI (green line in Figure 3-11B, 8 ± 3% in 120 s sample, 13 ± 3% in 30 min sample and 17 ± 3% in 6 hour sample). Given that she starting 56Fe MIOX(III/III) would not be seen, the detected MIOX(III/III)•MI is very likely due to a disproportionation of MIOX(II/III).

A B

Figure 3-11 4.2-K/53-mT Mössbauer spectra of MIOX samples. A. A solution of 2.62 mM MIOX(II/II) was 1:1 mixed at 7 oC with same concentration of MIOX(III/III) for 1 hour to obtain a sample. The samples of starting MIOX(II/II), MIOX(III/III) and the mixture after 1 hour were all added with MI before freezing for analysis. B. 2.23 mM 57Fe MIOX(II/II) was 1:1 mixed at 7 oC with same concentration of 56Fe MIOX(III/III) and treated with 100 mM MI after desired times. The color-coded solid lines overlaid with the data are the contributions of the MIOX(II/II)(•MI) (red), MIOX(II/III)•MI (blue) and MIOX(III/III)•MI (green).

Discussion

Alternative Mechanism for MIOX(II/II) Activation by O2

Since H2O2 was not detected with sensitive HRP assay in the reaction of

MIOX(II/II) with O2, the mechanism proposed in Scheme 3-1 is not favorable. In an alternative mechanism (Scheme 3-2) O2 is added to MIOX(II/II) and forms a peroxo

diiron(III/III), reminiscent of compound P in Methane Monooxygenase (MMO) (1,

58). The peroxo diiron(III/III) then reacts with another molecule of MIOX(II/II) and

Scheme 3-2 Kinetic mechanism for activation of MIOX(II/II) used in the simulations of Figure 3-8 (solid lines)a

aRate constants are estimates based on iterative simulation of all the data and visual evaluation of agreement. H+ to generate one equivalent of diiron(IV/III) and diiron(II/III) in close vicinity. In

E.Coli ribonucleotide reductase (RNR) R2 subunit, reaction of the reduced R2-D84E and R2-D84E/W48F mutants with dioxygen generates the peroxo diferric center (59,

60) and for the R2-D84E/W48F mutants, decay of the peroxo differic intermediate leads to intermediate X, a diiron(IV/III) center. The decay of X generates an oxidized oxo-bridged diferric center along with a stable unprotonated Tyr122•. Similarly, an intermolecular electron transfer between diiron(IV/III) and diiron(II/III) generates two equiv of diiron(III/III) species that was presumably detected in absorption and

Mössbauer spectroscopies. Subsequently, the produced two molecules of diiron(III/III)

comproportionate with two molecules of diiron(II/II) to produce four molecules of diiron(II/III). In this mechanism the diiron(IV/III) and diiron(II/III) are very close so the electron transfer can occur between them. If a small fraction of associated protein unit is dissociated to separated diiron(IV/III) and diiron(II/III), diiron(II/III) would become an earlier outcome than that produced by comproportionation, and diiron(IV/III) can continue to react with diiron(II/II) to form diiron(III/III) and diiron(II/III). Interestingly, the small fraction of dissociation of diiron(IV/III) and diiron(II/III) unit can account for the early formation of diiron(II/III) in the reaction of MIOX(II/II) with limiting O2 (Figure 3-9 and Figure 3-10). In the excess O2 reaction with MIOX(II/II), the slowly increasing MIOX(II/III) (Figure 3-5) can also be attributed to a small fraction of diiron(II/II) that does not react with O2 rapidly to form diiron(III/III) but produces small percentage of diiron(II/III). The kinetics of individual step has not been determined well to some extend due to the poor efficiency of the comproportionation. One caveat of this mechanism is the trapping of the peroxo diiron(III/III) intermediate. In R2-D84E and R2-W48F/D84E mutants (59,

60), MMOH (58, 61) and Δ9D (62), a similar peroxo-diferric core intermediate is formed and has a broad absorption band at 700 nm. This absorbance was not readily detected in MIOX(II/II) reaction with O2. One possible reason is that the unfavorable kinetics does not allow its accumulation.

This complex mechanism can still account for the result of MIOX(II/III) stoichiometry experiment in which MIOX(II/III) formation reaches its maximum when the value of O2:MIOX(II/II) ratio reaches 0.25 (Figure 3-1). The simulations

with the determined and estimated rate constants agree the result of [MIOX(II/III)] versus [O2]/[MIO(II/II)] stoichiometry experiment when [O2]/[MIOX(II/II)] is no larger than 0.25. When [O2]/[MIOX(II/II)] is larger than 0.25, our data suggest this mechanism is no longer applicable. MIOX(II/III) is still produced in a significant amount even though according to scheme 3-2 no MIOX(II/III) would be generated when [O2]/[MIOX(II/II)] is 0.5. The cause of this inconsistency has not been understood yet. One would think that a different mechanism might be relevant to physiologically oxidative stress and functions to mediate O2 response and protect the catalytically active form. In addition, one key difference of activation of MIOX from other binuclear non-heme iron enzymes is the lack of external reducing equivalents.

This plausible duo-function activation mechanism under various O2 environments could be delicately equipped to maintain mixed-valent MIOX(II/III) form for catalysis.

Implications of MIOX(II/II) Activation by O2

The activation of MIOX(II/II) to MIOX(II/III) by O2 is an unique process and has several key points to highlight. (1) the conversion of MIOX(II/II) to MIOX(II/III) is not a simple one-electron-oxidation; according to Scheme 3-2, four electrons required for O2 reduction to two H2O are from 4 molecules of MIOX(II/II), thus, the overall activation of MIOX(II/II) is actually a four-electron oxidation. (2) One molecule of

O2 can produce theoretical four molecules of active MIOX(II/III) state, which would assure in vivo the catalytically active form maximized under minimal O2

concentration if a same activation process occur in nature. (3) Comproportionation of

MIOX(II/II) with MIOX(III/III) involves a series of intermolecular electron transfer which requires the active sites of two molecules of protein in close vicinity. X-ray crystallography (39) shows MIOX is monomeric in solution and in the crystal and the diiron site is buried between two pairs of antiparallel helices, α4/α5 and α6/α7, and a fifth helix, α8. However the packing of the published structure (39) provides us a potential tunnel for electron transfer. The distance between two Fe1 sites is 36 Å and a number of residues in the middle probably function for proton-coupled electron-transfer (PCET) (Scheme 3-3). Such a long-distance (35 Å) PCET occurs in

Scheme 3-3 A possible tunnel between two molecules of MIOX and representative residues (cyan-red sticks) probably responsible for PCET. The distance is between two Fe1 sites of neighbor diiron centers (dark red balls). MI is shown as green-and-red- stick structure. the class I RNR reaction (63-65).

Implications of the Use of the Mixed-Valent Diiron Cluster

The use of the diiron(II/III) cofactor can rationalize several of the previously reported complex kinetic characteristics of MIOX. Most notably, the activation [both time-dependent activation and enhancement of activity in the steady state (20)] by thiols such as L-Cys, can be explained by their conversion of inactive MIOX(III/III) to active MIOX(II/III). Indeed, spectrophotometric monitoring of the conversion of the fully oxidized enzyme to the mixed-valent state by 5 mM L-Cys showed that the process is slow, requiring tens of minutes at 23 oC to approach completion (11). This time scale is consistent with the 5-10 min at 30 oC required for the time-dependent activation of the hog kidney enzyme (purified from its natural source) by L-Cys (20).

The use of the mixed-valent state also creates the possibility for redox regulation of

MIOX activity in physiological contexts. The established link between diabetic pathologies and oxidative stress (66) and the circumstantial links of derangements in

MI metabolism to these pathologies (67) make this possibility provocative.

Chapter 4 Dioxygenase Activity of myo-Inositol Oxygenase from Mus musculus with the Substrate Analogue 2L- 2,3,4,6/5-Pentahydroxycyclohexanone

The enzyme myo-inositol oxygenase (MIOX) catalyzes the oxidation of myo- inositol (MI) to D-glucuronate (DG) using O2 as the oxidant. This reaction entails cleavage of the C1-C6 bond (a two-electron reduction), and oxidation of C1 and C6 from the alcohol to the carboxylate (a four-electron oxidation) and aldehyde (a two- electron oxidation), respectively. This reaction proceeds with incorporation of one O- atom derived from O2 into the C1-carboxylate of the DG product, i.e. MIOX displays monooxygenase activity with the natural substrate, MI. The overall four-electron oxidation of MI is balanced by the four-electron reduction of O2 and therefore does not require a co-substrate. This unusual reaction begins with the addition of O2 to the mixed-valent Fe2(II/III) form of the substrate complex, MIOX(II/III)•MI, to generate a superoxo-Fe2(III/III) intermediate, termed G, that abstracts the C1 hydrogen from the bond. Subsequent steps in the reaction are less well understood. In this work, we have studied the reaction of the substrate analogue L-myo-inosose-6 (MI-6-one), in which –compared to the natural substrate, MI- the C6 carbon atom is oxidized from a secondary alcohol to the ketone. In the presence of MIOX(II/III), MI-6-one tautomerizes to the ene-diol form and gives rise to perturbations of the UV/visible-,

EPR-, and Mössbauer-spectroscopic features of MIOX(II/III). The MIOX(II/III)•MI-

6-one complex reacts with O2 to effect the four-electron oxidation of MI-6-one to the acyclic product 2,3,4,5-tetrahydroxy-hexane-1,6-dioate. The product is tentatively assigned to D-saccharate (DS, the product of C1-C6 cleavage), because it exhibits liquid-chromatographic and mass-spectrometric properties identical to those of a DS

18 standard. Experiments carried out with O2 reveal that the reaction proceeds with

partial incorporation of both O-atoms derived from O2. Thus, MIOX exhibits dioxygenase activity with the substrate analogue MI-6-one. The four-electron oxidation of a cyclic ene-diol moiety to the acyclic di-acid by MIOX(II/III) using O2 as oxidant is reminiscent of the reactions catalyzed by the ring-cleaving aromatic dioxygenases.

Introduction

Myo-inositol oxygenase (MIOX,1 EC 1.13.99.1) catalyzes the conversion of myo-inositol (MI) to D-glucuronate (DG, Scheme 4-1) (16, 68, 69), the first step in the only known pathway in humans for catabolism of MI (12), the sugar backbone of cell-signaling phosphoinositides (70). The reaction catalyzed by MIOX is a four- electron oxidation of the substrate (68). It entails cleavage of the C1-C6 bond (a two- electron reduction), oxidation of C1 from the alcohol to the carboxylate (a four- electron oxidation), and oxidation of C6 from the alcohol to the aldehyde (a two- electron oxidation). The overall four-electron oxidation of MI is balanced by the four- electron reduction of O2 and therefore does not require a co-substrate (57). The O- atom incorporated into the C1-carboxylate derives from O2, i.e. MIOX is a monooxygenase (68).

Although it has been known for nearly 50 years that MIOX requires iron (16), significant insight into its catalytic mechanism and its three-dimensional structure study have only emerged in recent years (9-11, 39, 57). We have shown by using a combination of UV/visible, EPR, and Mössbauer spectroscopies that MIOX harbors a

dinuclear non-heme-iron cofactor, which can exist in three oxidation states: Fe2(II/II),

Fe2(II/III), and Fe2(III/III) (9). These forms are referred to in the following as

Scheme 4-1 The natural substrate (MI) and substrate analogue (MI-6-one) reactions catalyzed by MIOX

MIOX(II/II), MIOX(II/III), and MIOX(III/III). Binding of MI significantly perturbs the spectroscopic features associated with MIOX(II/III) and MIOX(III/III), suggesting that MI may bind directly to the dinuclear cofactor. The X-ray structure of

MIOX reveals that MI binds directly to one of the Fe sites (this site is referred to in the following as Fe2) via its C1- and C6-bonded hydroxyl groups (39).

Compared to other dinuclear non-heme-iron oxygenases and oxidases, MIOX is distinct in many aspects. First, MIOX does not have significant sequence similarity to other non-heme-diiron proteins (39). In particular, MIOX lacks the two copies of the

ExxH motif, a hallmark feature of the non-heme-diiron proteins, of which the E and

H residues serve as ligands to the diiron cluster(40, 41, 71). Second, the architecture and fold of MIOX is different from that of the non-heme-diiron proteins (39). Third, the stoichiometry of the reaction is unusual, because MIOX catalyzes a four-electron oxidation.2(72)Fourth, and most importantly, the reaction mechanism is fundamentally different. The reaction of the non-heme-diiron oxygenases and oxidases begins with the addition of O2 to the fully reduced, Fe2(II/II) cofactor to generate a peroxo-Fe2(III/III) intermediate (1, 3, 4, 56, 73, 74). By contrast, the catalytically active form of MIOX contains the mixed-valent Fe2(II/III) cluster (9, 11), which reacts with O2 to generate a formally superoxo-Fe2(III/III) intermediate, termed

G, by addition of O2 to the Fe(II) site [which is presumably Fe1, based on the the X- ray structure (39)]and formally an inner-sphere electron transfer from the Fe(II) site to the O2 moiety (10, 26). G initiates the substrate oxidation by homolyzing the C1-H bond of MI. Evidence for this notion stems from the fact that G accumulates to much greater levels in the presence of the per-deuteriated MI substrate (the deuterium kinetic isotope effect was estimated to be 8-16) (10). Subsequent steps in the mechanism are not very well understood. Experimentally, a second intermediate state, termed H, has been trapped, but its identity has not yet been unraveled (11). Scheme

4-2 summarizes various possible mechanisms that lead to DG formation. These pathways diverge at the state generated by C1-H cleavage, which formally consists of a hydroperoxo-Fe2(III/III) complex and a C1-based substrate radical. Possible mechanisms include O-O homolysis to yield a C1-hemiketal intermediate and a

Fe2(III/IV) cluster (hydroxylation pathway) (10, 57). Loss of two electrons as shown

would yield the product, DG, and regenerate the Fe2(II/III) form. Alternatively, homolysis of the Fe-Ohydroperoxide bond would yield a C1-peroxyhemiketal

Scheme 4-2 Possible mechanisms for reaction MIOX(II/III)•MI with O2. Four possible reaction pathways (A - D) following cleavage of the C1-H bond by the formally superoxo-Fe2(III/III) intermediate, G, are depicted, which involve C1 hydroxylation coupled to peroxide O−O cleavage (pathway A), C1 hydroperoxylation by rebound of an • HOO radical (pathway B), C1 hydroperoxylation by addition of a second molecule of O2 to the C1 radical (pathway C) or electron transfer from C1 radical to the Fe site proposed by Morokuma (pathway D). intermediate and a Fe2(II/III) cluster (10, 57). Although Hamilton reported that non- enzymatic breakdown of the C1-peroxyhemiketal leads to DG (19), which suggests that this reaction pathway may be feasible, the rather large separation between C1 and the coordinated O-atom detected in the X-ray structure would make this reaction

pathway sterically less favorable. A sterically feasible adaptation of this mechanism may involve rebound via the distal O-atom of the (hydroperoxo) moiety to the C1- radical. This process may be coupled to translocation of the proton from the distal to the proximal O-atom. A third pathway, which was not considered previously, invokes addition of a second molecule of O2 to the substrate radical. In subsequent steps, the second molecule of O2 is fully reduced by four electrons. By contrast, the Fe1- coordinated (hydro)peroxide moiety would be reoxidized by two electrons to O2.

Thus, in this mechanism, O2 would serve two roles. The O2, which adds to the Fe1(II) site to generate G, is a cofactor, while the O2 molecule attacking the C1-centered substrate radical is a cosubstrate. Analogous roles for O2 have been reported for other enzymes. For the Mn-dependent enzyme oxalate decarboxylase, it was proposed that

O2 may serve the role as a cofactor (75, 76), while the addition of O2 to a carbon- centered radical is well known, e.g. in the Fe(II)- and α-ketoglutarate-dependent enzyme FtmOx1 (46) and the intradiol dioxygenases (77). The fourth pathway shown, which was proposed by Hirao and Morokuma, invokes a resonance form of the hydroperoxo-Fe2(III/III)•substrate radical intermediate: a hydroperoxo-Fe(II/III)•myo- inosose-1 intermediate (26). This resonance form results from inner-sphere electron transfer from the radical to the diiron cluster. In the Morokuma mechanism, nucleophilic attack of the distal O-atom of the coordinated (hydro)peroxo moiety on the C1-carbonyl of the myo-inosose-1 intermediate will lead to a bridging peroxyhemiketal intermediate, which is further converted upon O-O and C-C cleavage to the product, DG, and the resting Fe2(II/III) cluster (26). Although the

peroxo-Fe2(III/III) intermediates of the non-heme-diiron proteins do not exhibit nucleophilic character, the peroxo-metal complexes of several cytochrome P450 enzymes, the mononuclear non-heme-iron enzyme hydroxyethylphosphonate dioxygenase, and inorganic complexes are believed to attack carbonyl C-atoms as a nucleophile (38, 78-82) . Nucleophilic attack of a metal-bound peroxide on the carbonyl C-atom of the fatty aldehyde substrate was also proposed in the reaction catalyzed by cyanobacterial aldehyde decarboxylase (83).

It has been shown numerous times that the use of chemically or isotopically modified substrates may provide detailed insight into the mechanism of an enzyme reaction. In this work, we have used the analogue 2L-2,3,4,6/5- pentahydroxycyclohexanone (MI-6-one, Scheme 4-1), in which the C6 atom is oxidized from the secondary alcohol to the ketone (in comparison to the natural substrate, MI). We speculated that MI-6-one may undergo, in analogy to the natural reaction, C1-C6 cleavage and oxidation of C1 and C6. The product, in which C6 is oxidized by two more electrons (compared to the natural product, DG) would be the

2,3,4,5-tetrahydroxy-hexane-1,6-dioic acid, D-saccharate (DS, Scheme 4-1).

Consistent with our hypothesis, the reaction of the MIOX(II/III)•MI-6-one complex with O2 produces a stoichiometric amount of product that exhibits LC/MS properties

18 identical to those of an authentic DS standard. O2 tracer experiments further reveal that the reaction proceeds with incorporation of both O2-derived O-atoms, although one of them exchanges significantly (70%) with water. Thus, MIOX exhibits dioxygenase activity in its reaction with MI-6-one and O2. Although the outcome of

the reaction is analogous to that of the natural reaction, our data suggests that the mechanism is different. In the presence of MIOX(II/III), MI-6-one tautomerizes to the ene-diol form, which binds to the Fe2(II/III) cluster and perturbs its spectroscopic properties. The four-electron oxidation of an ene-diol involving cleavage of the C=C bond to the acyclic dicarboxylate is reminiscent of the reaction catalyzed by the intradiol dioxygenases (IDDs). IDDs are mononuclear non-heme-iron enzymes, which employ a high-spin Fe(III) site to coordinate and activate their substrate for subsequent reaction with O2 (84-86). The extradiol dioxygenases (EDDs) are related enzymes, which also cleave the C=C bond, but with a different regiospecificity(84, 85,

87). They are also mononuclear non-heme-enzyme, but employ a different mechanism, in which substrate coordinates the high-spin Fe(II) center, followed by addition of O2 to the Fe(II) center. The mechanisms of the EDDs and IDDs converge at the stage of a peroxyhemiketal intermediate (88). Because MIOX(II/III) harbors both a Fe(II) and a Fe(III) site, the mechanism may proceed by either reaction pathway. The peroxyhemiketal intermediate proposed to occur in the reaction with the MI-6-one analogue may be similar to that formed by nucleophilic attack of the distal O-atom of the coordinated (hydro)peroxide on the myo-inosose-1 intermediate in the native reaction proposed by Hirao and Morokuma (26).

Materials and Methods

Preparation of myo-inositol oxygenase (MIOX) reactant complexes.

myo-Inositol oxygenase (MIOX) from Mus musculus was overexpressed in and purified from Escherichia coli as previously described (9). Preparation of

MIOX(III/III) was carried out by addition of Fe(II)aq [the ammonium sulfate salt for natural-abundance Fe(II) and the sulfate salt prepared by dissolution of Fe(0) in

57 H2SO4 for Fe(II)] to a 30 μM solution of the iron-free MIOX protein at 5 ºC for 12 hours, followed by concentrating to ~3 mM. The purity of MIOX(III/III) is greater than 95% (determined by EPR and Mössbauer spectroscopies). MIOX(II/III) was prepared by comproportionation of a solution of 2 mM O2-free MIOX (III/III) with an equimolar amount of MIOX (II/II) [prepared as previously described (9)] for 4 hours at 7 ºC to yield a solution enriched in MIOX (II/III) (analysis of the solution presented in Figure 4-5). Preparation of the MIOX(II/III)•MI-6-one complex was carried out in an MBraun anoxic chamber by addition of MI-6-one (TCI America,

Portland, OR, > 98%) to MIOX(II/III) and incubation for two hours at 7 ºC. Details of the sample preparations are given in the figure legends.

Stopped-flow (SF) absorption-spectroscopic experiments

The SF apparatus has been described previously (11). In experiments to define the kinetics of substrate binding, MIOX(II/III) was mixed with an equal volume of an

O2-free solution of MI-6-one. To define the kinetics of the reaction of the

MIOX(II/III)•MI-6-one complex with O2, MI-6-one was added to a solution enriched in MIOX(II/III) to a concentration of 25 mM MI-6-one, and this solution was mixed

at 5 °C with an equal volume of buffer with variable O2 concentration. Reactant concentrations after mixing are given in the appropriate figure legend. Absorbance- versus-time traces from both binding of MI-6-one and reactions of MIOX(II/III)•MI-

6-one with O2 were analyzed by non-linear regression according to equation 1, in which k1 is apparent first-order rate constant, ΔA1 is amplitude for the exponential phase, and A0 is the absorbance at time zero.

(1)

For the binding of MI-6-one, the value of the apparent first-order rate constants as the function of concentration of MI-6-one were analyzed by a hyperbola fit according to equation 2, in which

λ is the apparent first-order rate constant, k-2 is the intercept with the y axis, k2 + k-2 is the asymptotic (maximum) rate constant, [S] is the concentration of MI-6-one and Kd is the dissociation constant (89).

Freeze-quench (FQ) EPR experiments

The apparatus and procedures for preparation of FQ EPR samples have been described previously (11). The same reactant concentrations were used in FQ experiment as SF. In the excess-O2 experiment (Figure 4-5B), the reaction solution was freeze-quenched after the desired time. The quoted reaction time for each sample is the sum of the known transit time through the reaction hose and the estimated

“quench time” (the time required for cooling to a temperature at with no further 93

reaction occurs) of 0.015 s. Following preparation of one of the samples, the reaction solution remaining in the hose was sealed inside the hose, incubated at 5 °C for 16.7 min, taken back into the anoxic chamber, transferred to an EPR tube, and frozen for

EPR analysis (bottom spectrum in Fig. 5B). The EPR spectrometer has been described previously (11). Spectral parameters are given in the figure legends.

Mössbauer spectroscopy

The spectrometer has been described previously (9). Specific conditions are given in the appropriate figure legend. Simulation of Mössbauer spectra was carried out using WMOSS (WEB Research, Edina, MN). Some of the simulations are based on the spin Hamiltonian formalism, given by equation (3), in which the first term describes the electron Zeeman effect, the second term represents the interaction between the electric field gradient and the nuclear quadrupole moment, the third term describes the magnetic hyperfine interactions of the electronic spin with the 57Fe nuclei, and the last term represents the nuclear Zeeman interaction.

⦁ ⦁

⦁ ⦁ ⦁ (3)

Simulations were carried out with respect to the total electronic spin of the ground state (S =1/2) with g-values known from EPR. The hyperfine tensors with respect to the total spin, A, are related to the intrinsic hyperfine tensors, a, by the

following relation: Ai = ci ai, where c1 = +7/3 for the Fe(III) site (S1 = 5/2) and c2 = -

4/3 for the Fe(II) site (S2 = 2).

Liquid chromatography/mass spectrometry (LC/MS) analysis

A 10 µL aliquot of sample was injected onto a PRX-X300 anion exclusion column (250 mm × 4.1 mm, 7 µm particle size; Hamilton, Reno, NV) in 0.05% triethylamine / 50% methanol / 50% water mobile phase. The column was developed isocratically at a flow rate of 0.2 mL/min. The eluate was passed through a Waters

(Milford, MA) MicroMass ZQ 2000 mass spectrometer operating with electrospray ionization in the negative-ion mode. Total ion monitoring at m/z values from 5 to 300 was performed during elution, and the intensity of different peak was determined. For quantitation of the product (presumed to be DS), the reaction of MIOX(II/III)•MI-6-

18 one was carried out with limiting amount of O2, quenching of the reaction by heat denaturation for 3 min at 85 ºC, addition of a known amount of unlabeled DS, and analysis by LC/MS [single-ion monitoring in the range of m/z 209 to 213 of product peak (retention time of 13.1 min)]. Comparison of the peak area of the amount of unlabeled DS (m/z = 209) to that of enzymatically produced DS (m/z = 211 and 213) allows the amount of enzymatically produced DS to be determined.

Determination of Kd for binding of MI-6-one to MIOX(II/III)

In an MBraun anoxic chamber, aliquots of O2-free solution of 0.24 mM enriched MIOX(II/III) were mixed with varying amounts of a MI-6-one stock

solution (610 mM or 61 mM) and incubated for two hours at 7 oC. The solutions were transferred to EPR tubes and frozen in liquid nitrogen for spectroscopic analysis. The concentrations are given in the figure legends. The intensity (I) of the EPR signal associated with the MIOX(II/III)•MI-6-one complex and MIOX(II/III) was obtained from the peak-to-trough distance at the g-value of 1.78. The intensities were corrected for the contribution of the signal from the spectrum of MIOX(II/III) at that g-value.

Fractional occupancies of enzyme at different concentration of MI-6-one were derived by equation (4):

Occupancy = [IMIOX(II/III)•MI-6-one - IMIOX(II/III)] / IMIOX(II/III)•MI-6-one, max (4)

The fractional occupancies of enzyme as the function of concentration of MI-6-one were analyzed with a quadratic equation 5, in which θ is the fractional occupancy, A0 is the concentration of MI-6-one, Kd is the dissociation constant, A1 is the maximum of the amplitude and A2 is the intercept on the y axis. The total concentration of

MIOX(II/III) was held fixed at the value known from EPR analysis.

Tautomerization of MI-6-one in D2O in the presence of MIOX(II/III)

The isotope exchange of MI-6-one upon binding to MIOX (II/III) was analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). The binding reactions were carried out in deuterium-enriched buffer [50 mM Bis-Tris-acetate (pH

6.0) and 10% (w/w) glycerol (OD)3 (98%) in D2O (99.9%)] in an MBraun anoxic 96

chamber. Replacement of the exchangeable protons of the MIOX by deuterium was achieved in the following manner. At 4 °C, purified apo-MIOX and reconstituted

MIOX(III/III) were loaded on a Centriprep YM-10 centrifugal filter unit (Millipore,

Bedford, MA) respectively. Minimum dilutions with buffer followed by concentration were carried out 10 times in total. All enzyme, substrate, buffer were deoxygenated prior to mixing in the anoxic chamber. To a solution containing 1.1 mM MIOX(II/III) was added at 5 °C limiting MI-6-one to give a final concentration of 0.9 mM MI-6-one, and, at desired times the samples were incubated at 85 °C for 3 minutes to inactivate the enzyme. After centrifugation to pellet the denatured protein, water was removed in vacuo from the supernatant using a SpeedVac concentrator

(Thermo Fisher, Asheville, NC), and the dried fraction was dissolved in the mobile phase for subsequent analysis by LC/MS. The sums of the intensities of unlabeled

MI-6-one with m/z = 177 and those of deuterium-enriched MI-6-one with m/z = 178 were normalized to 1 thus the relative intensity was derived for individual peak.

18 Reactions of MIOX(II/III)•MI and MIOX(II/III)•MI-6-one in the Presence of O2(g)

All experiments were carried out in the anoxic chamber. An O2-free solution of

MIOX(II/III) [0.5 mM MIOX, 0.4 mM Fe, 65% enrichment in mixed-valent state] and MI-6-one (50 mM) was divided into three aliquots. Two aliquots were sealed in

18 test tubes. . One aliquot in tube was subsequently mixed with 52 µL O2 (g) (99% isotopic enrichment, ICON, Marion, NY)-saturated buffer to give a final

18 concentration of 0.2 mM MIOX(II/III), 50 mM MI-6-one and ~ 0.17 mM O2, and a

16 second aliquot was mixed with natural-abundance O2-saturated buffer to give

18 identical final concentrations as O2 reaction. The reaction solutions were incubated for 30 min at 5 °C to ensure the reactions to reach completion, and placed at 85 °C for

3 minutes to inactivate the enzyme. To the third aliquot of MIOX(II/III)•MI-6-one solution (the control) 26 µL O2-free buffer was added and incubated for 30 min in the anoxic chamber followed by heat inactivation and centrifugation. To quantify the product in the reaction of MIOX(II/III)•MI-6-one with O2, each reaction solution

18 16 ( O2 or O2) was divided into two equivalent aliquots. One aliquot was applied directly onto centrifugation and a second aliquot was added with known amount of

DS as internal standard followed by centrifugation. The supernatants after centrifugation were subjected to LC-MS analysis as described above.

Results

UV/Visible-Spectroscopic Perturbation upon MI-6-one Binding

Addition of MI-6-one to a solution enriched in MIOX(II/III) results in decrease in the absorption at 495 nm and an increase in absorption in the ~750 nm region

(Figure 4-1). The asymmetric shape and large width of the new low-energy features suggest the presence of multiple, overlapping absorption bands. The difference spectrum obtained upon addition of MI-6-one (Figure 4-1, inset) displays a distinct minimum at ~495 nm, corresponding to absorption from MIOX(II/III) that is lost

upon binding of MI-6-one, and a maximum at ~750 nm corresponding to absorption from the developing

MIOX(II/III)•MI-6-one complex(es). The new absorption features are tentatively assigned to low-energy charge-transfer Figure 4-1 UV/visible absorption spectra transitions of the analogue MI-6-one in its of MIOX(II/III) in the absence and ene-diolate form (see below) coordinating presence of MI-6-one. A solution of 0.5 mM total MIOX enriched in the Fe(III) center of MIOX(II/III) in a MIOX(II/III) (0.24 mM, 1.6 eq. of Fe, bidentate fashion. Similar low-energy red) was mixed with 73 mM MI-6-one to give a final 0.2 mM MIOX(II/III) and catecholate-to-Fe(III) transitions have incubate for 2 hrs (at 7 °C, blue). been observed for the IDDs (90, 91).

Evidence for Tautomerization of MI-6-one upon Binding to MIOX(II/III)

The development of the low-energy absorption features suggest that MI-6-one may bind in its ene-diol form as a bidentate ligand to the Fe(III) site of MIOX(II/III).

Because the proposed tautomerization entails cleavage and re-formation of the C1-H bond, we have monitored the incorporation of deuterium into unlabeled MI-6-one from D2O solvent as a function of the incubation time. The mass spectrum of unlabeled MI-6-one exhibits its molecular ion peak at m/z = 177.2 in negative ion mode (Figure 4-2, t = 0), which corresponds to the singly deprotonated form of MI-6-

one. The intensity of the (M+1) peak at m/z = 178.2 (6% of that of the peak with m/z

= 177.2) agrees well with the calculated intensity (6.6 %). When unlabeled MI-6-one is incubated with apo MIOX for varying reaction times and analyzed for the deuterium incorporation by MS, the intensity ratio of the peaks at m/z = 177.2 and m/z

Figure 4-2 Mass spectra depicting hydrogen exchange of MI-6-one in presence of MIOX(II/III). In a deuterium-enriched solution, 1 mM MI-6-one was incubated with

either O2-free buffer or MIOX(II/III) (2.4 mM MIOX, 1.6 eq. of Fe, prepared as described in Materials and Methods, middle) for one hour. Heat-denatured protein was removed by centrifugation. The deuterated buffer of the supernatant was exchanged with protium-enriched buffer to wash out exchangeable deuteria. Representative relative intensity shows that MI-6-one (m/z = 177.2, green) gains one more mass (m/z = 178.2, blue) when mixing with MIOX(II/III).

= 178.2 does not change over time (data not shown). By contrast, when limiting (0.9 mM) MI-6-one is incubated with excess (1.2 mM) MIOX(II/III), the intensity of the peak at m/z = 177.2 decreases with time at the expense of the peak at m/z = 178.2

(Figure 4-2). The increase in intensity of the peak at m/z = 178.2 can be rationalized by generation of an MI-6-one isotopolog, in which a single H-atom has been

100

exchanged with deuterium from solvent.3 This result supports tautomerization of MI-

6-one to the ene-diol form in the presence of MIOX(II/III).

EPR-spectroscopic Perturbation upon MI-6-one Binding

In the absence of substrate,

MIOX(II/III) exhibits a very broad, axial signal in X-band EPR with effective g- values of 1.95, ~1.66, and ~1.66 (Figure

4-3, top), which emanates from the antiferromagnetically (AF) coupled, Figure 4-3 EPR spectra of MIOX(II/III) valence-localized, high-spin Fe2(II/III) in the absence and presence of MI-6- one. A solution of 0.5 mM total MIOX cluster with S = 1/2 electron spin ground enriched in MIOX(II/III) (0.24 mM, 1.6 state (92, 93). Addition of a saturating eq. of Fe, top) was mixed with 73 mM MI-6-one to give a final 0.2 mM amount of MI-6-one (73 mM, see below) MIOX(II/III) and incubate for 2 hours to a sample of MIOX(II/III) results in a (at 7 °C, bottom). significant perturbation of the EPR spectrum (Figure 4-3, bottom). The spectrum of

MIOX(II/III)•MI-6-one displays multiple features with effective g-values of less than

2. The spectrum suggests the presence of more than one Fe2(II/III)-containing species.

The spectrum is much narrower than that of MIOX(II/III), which suggests that MI-6- one binding is associated with a significant perturbation of the electronic structure, most likely involving a strengthening of the exchange coupling between the Fe(II)

101

and Fe(III) sites upon binding of MI-6-one. A similar effect is observed for binding of the natural substrate, MI, to MIOX(II/III) (9).

Mössbauer-spectroscopic Perturbation upon MI-6-one Binding

As a prerequisite for our Mössbauer-spectroscopic studies on the effect of MI-6- one binding to MIOX(II/III), we have analyzed Mössbauer spectra of a sample enriched in MIOX(II/III) as described in Materials and Methods using the reference spectra reported before for MIOX(II/III) and MIOX(III/III) (9) and simulated spectra for MIOX(II/II) because the heterogeneity of MIOX(II/II) leads unrepeatable spectra.

The analysis reveals that the sample contains 60 ± 8 % of MIOX(II/III), 11 ± 4 %

MIOX(III/III), and 29 ± 4 % of MIOX(II/II) (Figure 4-4). Addition of a saturating amount of MI-6-one (50 mM) to the above sample results in significant

Figure 4-4 Mössbauer spectra of MIOX(II/III). A sample of 57Fe-enriched MIOX(II/III) prepared by the comproportionation method described in Materials and Methods was recorded at 4.2 K in a 53-mT external magnetic field oriented parallel to the propagation direction of the γ-beam (top) and at 120 K without applied magnetic field (bottom). Solid lines above the data are the contributions of MIOX(II/II) (red, 29 ± 3%), MIOX(II/III) (blue, 60 ± 5%), and MIOX(III/III) (green, 11 ± 3%). The solid black lines overlaid with the experimental data (vertical bars) represent the added contribution of the three components.

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Figure 4-5 Mössbauer spectra of MIOX(II/III)•MI-6-one A sample of 57Fe subject to the comproportionation method described in methods and materials and then treated with 50 mM MI-6-one. A and B: 4.2-K/53-mT Mössbauer spectra of MIOX(II/III)•MI-6-one with parallel and perpendicular externally applied field (indicated on the top). C: 120-K/zero-field Mössbauer spectra of MIOX(II/III)•MI-6- one. Top spectra show raw data (hashed marks) and the contribution of MIOX(II/II)•MI-6-one (28% of the total intensity, solid line in red) and MIOX(III/III)•MI-6-one (9% of the total intensity, solid line in green). Removal of their contributions yields the reference spectra of MIOX(II/III)•MI-6-one (middle in A, bottom in B and C). The solid line overlaid with the data is a simulation according to the parameters from Table 1, and the solid and dotted lines plotted above the data are the contributions of the Fe(II) and Fe(III) sites, respectively. perturbation of the Mössbauer spectra (Figure 4-5). Analysis of spectra recorded at

4.2 K in a 53-mT magnetic field oriented either parallel (Figure 4-5A) or perpendicular (Figure 4-5B) to the propagation direction of the γ-beam and recorded at 120 K without applied field (Figure 4-5C) reveals that the sample contains 28 ± 6 %

MIOX(II/II)[•MI-6-one], 9 ± 4 % MIOX(III/III)•MI-6-one, and 62 ± 8 %

MIOX(II/III)•MI-6-one.4 The contribution of MIOX(III/III)•MI-6-one and

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Table 1 Mössbauer Parameters of MIOX(II/III)•MI-6-one and MIOX(II/III)•MI (italicized

values are parameters generated from 120-K/zero-field spectra)

a a a δ (mm/s) ∆EQ (mm/s) ƞ A/gNβN (T) percentage

MIOX(II/III)•MI-6-one

Fe(III) site in MIOX(II/III) 0.49 -0.8 0.5 (-45.7, -56.6, -52.0)

0.52 0.87 33%

Fe(II) site in MIOX(II/III) 1.22 2.41 0.2 (24.1, 21.8, 12.8)

1.17 2.41 33%

Fe(III) site in MIOX(III/III) 0.55 0.94

0.52 0.70 9% Fe(II) site in MIOX(II/II) 1.28 3.12

1.17 2.41 25%

MIOX(II/III)•MI

Fe(III) site in MIOX(II/III) 0.49 -1.11 7.2 (-43.1, -55.8, -53.4)

Fe(II) site in MIOX(II/III) 1.12 2.68 0.3 (26.5, 22.5, 11.6)

MIOX(II/II)[•MI-6-one] is modeled by two quadrupole doublets with the parameters given in Table 1 (green and red lines in top spectra). Removal of the contribution of

MIOX(II/II)•MI-6-one and MIOX(III/III)•MI-6-one yields reference spectra of

MIOX(II/III)•MI-6-one (middle spectrum in 4-5A and bottom spectra in 4-5B and 4-

5C). These spectra fully support the notion that MIOX(II/III)•MI-6-one harbors a valence-localized high-spin Fe2(II/III) cluster. At 120 K in the absence of a magnetic field, the paramagnetic features collapse to quadrupole doublets with parameters typical of high-spin Fe(III) and Fe(II) sites (Figure 4-5C, bottom). The 4.2-K/53-mT reference spectra of MIOX(II/III)•MI-6-one, as well as the parallel-minus- 104

perpendicular difference spectrum (Figure 4-5A, bottom), are typical of a valence- localized, high-spin Fe2(II/III) cluster in the slow relaxation limit and can be simulated according to the spin Hamiltonian formalism. Simulations with parameters given in Table 1 are shown as solid lines overlaid with the reference spectra, and the individual contributions of the Fe(II) and Fe(III) sites are plotted above the data as solid and dotted lines, respectively. We would like to note that the large number of parameters required for simulation of the magnetically split spectra of

MIOX(II/III)•MI-6-one does not allow for their unambiguously determination.

Nevertheless, the parameters fully support the presence of this form of the enzyme.

Kinetics of MI-6-one Binding to MIOX(II/III) and Determination of Kd

The intensity of the EPR signal associated with the MIOX(II/III)•MI-6-one complex increases with increasing [MI-6-one] and approaches saturation upon addition of > 25 mM MI-6-one (Figure 4-6). Plots of the fractional occupancy of

MIOX(II/III) as a function of the concentration of MI-6-one (inset of Figure 4-6) were analyzed to give an apparent K1k2 of 0.52 ± 0.09 mM. The kinetics of binding of

MI-6-one to MIOX(II/III) can be monitored by the increase of absorbance at 720 nm

(∆A720) (Figure 4-7). The kinetic traces at 720 nm were analyzed with a single exponential term. A plot of the apparent first-order rate constant obtained from this analysis versus [MI-6-one] can be analyzed with a hyperbola (Figure 4-7, inset) which indicates a two-step mechanism for generation of the chromophore. This

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Figure 4-6 Titration of 0.24 mM MIOX (II/III) with MI-6-one by EPR spectroscopy. The spectra shown correspond to 0 mM (red), 0.3 mM (blue), 0.9 mM (green), 2.7 mM (yellow), 24.3 mM (dark) and 72.9 mM (orange) MI-6-one. The points in the inset depict the occupancy of MIOX(II/III) as a function of MI-6-one concentration. The solid line is a fit of the quadratic equation for binding to the data, yielding K1k 2= 0.52 ± 0.09 mM. behavior can be rationalized by binding of MI-6-one in the keto-form to the enzyme, perhaps as shown in Scheme 4-4 in a monodentate fashion to the Fe(III) site with low affinity (1/K1 = 50 mM). Tautomerization to yield the ene-diol(ate) form is then proposed to result in tighter binding (K1k2 = 0.52 mM) to the Fe(III) site in a bidentate fashion and development of the low-energy charge-transfer transition in the absorption spectrum.

Figure 4-7 Kinetics of absorbance at 720 nm after mixing (at 5 °C) of a solution enriched in MIOX(II/III) (0.2 mM) with an equal volume

of an O2-free solution of MI-6-one to give a final MI-6-one concentration of 0.5 mM (o), 1.5 mM (□), 5 mM (◊), 15 mM (×) and 50 mM (+). The solid lines are fits of the equation for single first-order decay process to the data. The inset is a plot of the rate constant versus [MI-6-

one] with a hyperbola fit, yielding Kd = 52 ± 4.4 mM.

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Reaction of MIOX(II/III)•MI-6-one with O2

The reaction of the MIOX(II/III)•MI-6-one complex with excess O2 was investigated by SF-absorption and FQ-EPR spectroscopies. The reaction results in rapid loss of the ~720 nm (maximum wavelength measured by stopped-flow) absorption feature, implying that the MIOX(II/III)•MI-6-one complex is consumed, and an increase in absorption in the ~ 495 nm region (Figure 4-8A), which is characteristic of free MIOX(II/III), and implies that product has been formed and released. The time dependence at these two wavelengths shows a separation between the development of the negative 720 nm and positive 495 nm features (Figure 4-8C), which implies that more events would occur between MIOX(II/III)•MI-6-one and

MIOX(II/III). At the longest reaction time that SF was able to detect (1000 s), the spectrum was nearly identical to the first reliable spectrum (0.025 s), suggesting that the starting MIOX(II/III)•MI-6-one complex is regenerated. Thus the reaction is catalytic. The slow formation phase at 720 nm with the apparent first-order rate constant of 1.5 × 10-3 s-1 is consistent with the kinetics of MI-6-one binding to

MIOX(II/III) [1.9 × 10-3 s-1 when the concentration of MI-6-one is 15 mM]. Similarly,

EPR spectra of samples prepared by the FQ method confirm that MIOX(II/III)•MI-6- one reacts rapidly with O2 and is subsequently regenerated (Figure 4-8B). Spectra of samples freeze-quenched during the reaction show that the signal of the reactant complex decays rapidly and redevelops slowly with kinetics (Figure 4-8C, circles) similar to those reflected by the absorbance changes at ~720 nm in the SF experiment carried out under comparable conditions (Figure 4-8C, blue line). The reaction of

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B C

Figure 4-8 Kinetics of the reaction of the reaction of MIOX(II/III)•MI-6-one complex with excess O2. A sample of the MIOX(II/III)•MI-6-one complex (1 mM MIOX, 1.6 equiv of Fe, 50 mM MI-6-one) prepared as described in Materials and Methods, was mixed at 5 °C with an equal volume of the O2 saturated (at 5 °C) buffer to give a final

0.28 mM MIOX(II/III), 25 mM MI-6-one, and 0.71 mM O2. The reaction was analyzed by both optical absorption (panel A) and X-band EPR (panel B) spectroscopies. (A) The spectra reflect the reaction times of 0.025 s (blue), 0.05 s (red), 0.10 s (green), 0.20 s (brown), 10 s (cyan) and 1000 s (black). (B) The samples were rapidly frozen at the indicated reaction times. The signal intensity of MIOX(II/III)•MI-6-one (obtained as described in Materials and Methods, black circles in panel C) is scaled for direct comparison to the absorbance change. Spectrometer conditions were as follows: temperature, 10 K; microwave frequency, 9.45 GHz; microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 10 G; time constant, 327 ms; scan time, 167 s. (C) The curve shows the kinetics of absorbance at 495 nm (red line) and 722 nm (blue line) which was directly compared to the EPR signal intensity. (D) Kinetics of reaction of the MIOX(II/III)•MI-6-one complex with excess O2. A solution of the MIOX(II/III)•MI-6-one complex [0.2 mM MIOX(II/III), 50 mM MI-6-one] was mixed at o 5 C with an equal volume of an O2 soluiton. The final O2 concentrations are 0.18 (blue ○), 0.24 (red □), 0.36 mM (green ◊) and 0.71 (black Δ) mM. The solid lines are fits according to eq 1. The inset shows kobs values for the decay phase of the reaction vs. O2 concentration. The plot gives second-order rate constant of (49 ± 2) × 103 M-1 s-1.

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3 -1 -1 MIOX(II/III)•MI-6-one with O2 is only two fold slower [(49 ± 2) × 10 M s , Figure

4-8D], compared to the reaction with the natural substrate [1 × 105 M-1s-1 (11)].

However, no species with distinct EPR features accumulates to detectable amounts on the pathway to the product and MIOX(II/III). This observation seems to contradict the stopped-flow data, which suggests at least one intermediate between

MIOX(II/III)•MI-6-one and MIOX(II/III). This can be rationalized in the following ways. First, the intermediate may be EPR-inactive and is therefore not detected by

FQ-EPR spectroscopy. This scenario is disfavored on the basis of our proposed mechanism (Scheme 4-4) in which all species of the various possible mechanisms have a half-integer electron spin ground state and are therefore expected to be EPR- active. Alternatively, the EPR-spectroscopic properties of the intermediate might not be distinct from those associated with either the starting MIOX(II/III)•MI-6-one complex or MIOX(II/III).

Characterization of the Product of the Reaction of MIOX(II/III)•MI-6-one with O2 –

Evidence for Dioxygenase Reactivity of MIOX(II/III)

The transient-state spectroscopic data presented in the previous section shows that the MIOX(II/III)•MI-6-one complex reacts rapidly with O2 and is subsequently regenerated. We speculate that, in analogy to the natural reaction, MI-6-one is oxidized by four electrons to the acyclic product with the C1-C6 bond cleaved, D- saccharate (DS, see Scheme 4-1). We have tested this hypothesis by using LC/MS to

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analyze for the product of the reaction. The chromatogram (monitored in negative ion mode for m/z values ranging from 5 to 300) exhibits two major peaks (Figure 4-9).

The peak with a retention time of 20.8 min is assigned to the reactant, MI-6-one, based on the control experiment, in which a MI-6-one solution was examined under identical conditions. The second peak, which elutes with a retention time of 13.1 min, is assigned to the product of the reaction. Consistent with our hypothesis that the product of the reaction is 2,3,4,5-tetrahydroxy-hexanedioate (presumably DS), a solution of DS elutes with a retention time of 13.1 min (Figure 4-9). Further insight

Figure 4-9 High performance liquid chromatogram mass spectrometry (HPLC-MS) analysis of the commercial MI-6-one and DS upon its incubation in 50 mM Bis-Tris- acetate, 10% (w:w) glycerol buffer (pH 6.0). MI-6-one (blue) and DS (red) were dissolved to 0.025 mM and 0.3 mM respectively in the buffer and analyzed by HPLC-MS, as described in the Material and Methods. The traces shown are of the intensity of negative- ion monitored at m/z 209 (M- for DS) and m/z 177 (M- for MI-6-one). Note that the peak centered at 23 min for m/z 209 is from the natural abundant 13C isotopologue of Bis-Tris (m/z = 208) in buffer which is dominated (50 mM) in the sample and were not seen in the standard DS sample dissolved in water. into the reaction between MIOX(II/III)•MI-6-one with O2 was obtained by analyzing mass spectra generated by integrating peaks for selected m/z values in the range from

110

209 to 213 (Figure 4-10). In the anaerobic control sample, the intensity of the product peaks (red bars in Figure 4-10A) is much less than those observed for samples exposed to O2. The low intensity at m/z = 209, which is indicative of product formation, in the anaerobic control is presumably due to low amounts of contaminating O2 in the anoxic chamber. The contribution of the anaerobic control was subtracted from the peak intensities of samples, in which the MIOX(II/III)•MI-6- one complex is exposed to a limiting amount of O2. The integrated intensities of the product peak in the chromatogram for different m/z values provide insight into the reaction mechanism. When MIOX(II/III)•MI-6-one is mixed with unlabeled O2 (>99%

16O, Figure 4-10B, cyan bars), the integrated intensity is greatest for the peak at m/z =

209. The Δ(m/z) value of +32 (compared to the substrate peak at m/z =177) suggests the incorporation of two O-atoms into the product. The intensity of the peak at m/z =

210 is 7.2% of that of the peak at m/z = 209, which is close to the value expected for

18 the isotopologs with Δ(m/z) = +1 (6.6 %). When the reaction is carried out with O2

(>98% 18O, Figure 4-10B, blue bars), the most intense peak is observed at m/z = 211.

16 The Δ(m/z) = +2 (compared to the major peak of the O2 experiment) suggests incorporation of one 18O atom into the product. The peak at m/z = 213 emanates from product with two 18O atoms incorporated and provides direct evidence for dioxygenase activity of MIOX(II/III) towards MI-6-one. The lower intensity (42% of that of the peak at m/z = 211) reveals that one of the two O-atoms exchanges with

18 solvent. The intensity of the peak at m/z = 209 in the O2 experiment is only 19% of

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A B C

Figure 4-10 Mass spectra depicting intensities (A and B) and concentrations (C) of 16 products in the reaction of MIOX(II/III)•MI-6-one with either O2-free buffer (red), O2– 18 containing buffer (cyan and yellow), or O2-containing buffer (blue and green). In a sample, the MIOX(II/III)•MI-6-one complex prepared as described in Materials and 16 18 Methods was mixed with O2-free buffer (red), O2-saturated buffer or O2-saturated buffer subsequently followed by incubation at 5 °C for 30 min. Each oxygented solution was divided into two identical aliquots. An aliquot directly proceeded to heat-denaturing step (blue for 18O or cyan for 16O). A second aliquot solution was added with 0.15 mM DS as internal standard and followed by heat-denaturing (green for 18O or yellow for 16O). After removal of heat-denatured protein by centrifugation, the supernatant was subjected to the LC-MS for product analysis. A: The raw peak intensity of products from the reactions. B: The intensity of peak in anaerobic control sample was removed from that of each corresponding peak in the reaction samples. C: The concentration of 18 16 productsin the sample mixed with O2 (blue) or O2 (cyan) was obtained by comparing the peak intensity of product (m/z = 209-214) with that of 0.15 mM DS (m/z = 209) (see result section in text). that at m/z = 211. This observation can be rationalized either by a small amount of

16 18 contaminating O2 or a low, but detectable washout of both O atoms, or both.

To quantify the product of the reaction, the experiments were repeated under identical conditions except for addition of 0.15 mM unlabeled DS as an internal

18 standard (Figure 4-10B, green and yellow bars for experiments carried out with O2

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16 and O2, respectively). The intensity difference between the samples with and

18 16 without addition of DS in either O2 or O2 reaction accounts for 0.15 mM unlabeled

DS. Subsequently the intensities of all peaks can be converted to the concentrations of

18 products (Figure 4-10C). In the reaction with O2 (blue bars in Figure 4-10C), ~0.13 mM single 18O incorporated DS (m/z = 211) and ~0.06 mM double 18O incorporated

DS (m/z = 213) are formed. By the contrast, ~0.15 mM unlabeled DS (m/z =209) is

16 the major product in the reaction with O2 (cyan bars in Figure 4-10C). Given that

~0.17 mM O2 was reacted with excess MIOX(II/III)•MI-6-one complex, this reaction exhibits a product:O2 stoichiometry or 1 (within the limits of the method).

Discussion

In this work we have studied the mechanism of the reaction between O2 and the active form of myo-inositol oxygenase, MIOX(II/III), with MI-6-one, the substrate analogue of myo-inositol, MI, in which the C6-atom is oxidized from the secondary alcohol to the ketone. We speculated that (in analogy to the MIOX(II/III)-catalyzed conversion of MI to DG) the four-electron oxidation of MI-6-one might entail cleavage of the C1-C6-bond and yield the acyclic 1,6-diacid, D-saccharate, DS. When considering the tautomeric ene-diol form of MI-6-one, this four-electron oxidation of the substrate formally entails cleavage of a C=C bond (a four electron reduction) and oxidation of the two C-atoms from the alcohol to the acid (two four-electron oxidations). This outcome is remarkably similar to that of the intradiol dioxygenases

(IDDs), which catalyze the conversion of catechol to cis-cis-muconic acid (Scheme 4-

3A). IDDs belong to the large family of mononuclear non-heme-iron enzymes. Their 113

mechanism has been studied in great detail by a combination of biochemical, kinetic, spectroscopic, and crystallographic methods (94-97). The active form of IDDs harbors a high-spin Fe(III) center that binds the catechol substrate and gives rise to low-energy (λmax ~600 nm) catecholate-to-ferric CT bands (90). O2 adds to a C-atom of the ene-diol moiety, due to the partial semiquinone character of the Fe(III)- catecholate complex. Next, the distal O-atom attacks the Fe(II) center and forms a peroxyhemiketal complex, which undergoes the Criegee rearrangement to form the anhydride of the muconic acid and a Fe(III)-OH complex. Hydrolysis of the anhydride finally yields the product, muconic acid. Isotope

Scheme 4-3 Reactions catalyzed by the interdiol dioxygenases (A) and extradiol dioxygenases (B).

18 labeling studies with O2 demonstrate that both O2-derived oxygens are incorporated into the product, but one of the labels is partially washed out by solvent (98).

The extradiol dioxygenases (EDDs) are also mononuclear non-heme-iron enzymes that cleave the C=C bond of a catechol substrate, but they cleave the ring adjacent to the C(OH)=C(OH) fragment (Scheme 4-3B) (86, 87, 99). Detailed studies of their reaction mechanisms reveal that their mechanism is distinct from that of the

IDDs. The EDDs use a high-spin Fe(II) center to bind the catecholate substrate and activate O2 at the Fe(II) center (rather than the semiquinonate substrate as in the IDDs) to yield a Fe(III)-superoxo intermediate, which in the next step attacks the C-atom of 114

the ene-diol moiety to form the peroxyhemiketal intermediate (100). Criegee rearrangement, followed by hydrolysis of the anhydride will then yield the product(101, 102). As the IDDs, EDDs are dioxygenases and incorporate both O- atoms from O2 into the substrate, albeit one of the labels is partially washed out with solvent (103).

Although the outcome of the reaction of MIOX(II/III)•MI-6-one with O2 is similar to that of the IDDs, multiple mechanistic pathways are conceivable (Scheme

4-4). Because the active form of MIOX(II/III) harbors both a Fe(II) and a Fe(III) site, the possible mechanisms incorporate elements of the IDDs and EDDs. Two lines of evidence suggest that MI-6-one binds to MIOX(II/III) in its ene-diolate form. First, binding of MI-6-one gives rise to low-energy absorption feature(s) at ~750 nm.

Similar features have been observed in the IDDs and been assigned to catecholate-to-

Fe(III) CT transitions (90). Binding of MI-6-one to the Fe(III) site of MIOX(II/III) can be reasonably expected, because the natural substrate, MI, binds to the Fe(III) site, and the hydroxyl groups on C2, C3, C4, and C5 of MI-6-one have the same stereochemistry as those in MI and are likely to engage in the same hydrogen bonding interactions with Asp-85, Ser-87 and Asp-142 in the active site (39). Second,

MIOX(II/III) promotes the tautomerization of MI-6-one, as shown by a Δ(m/z) of +1 in D2O, which suggests the incorporation of one deuterium into MI-6-one. The kinetics of MI-6-binding are complex and suggest that it occurs in multiple steps, as was observed for the IDDs(48, 104, 105). We depict substrate binding in sequential steps, in which MI-6-one may coordinate the Fe(III) initially as a monodentate ligand

115

and, upon tautomerization, can serve as a bidentate ligand. As for the IDDs, two resonance structures are conceivable: the Fe2(II/III)-ene-diolate and Fe2(II/II)-ene-

Scheme 4-4 Possible mechanisms for the reaction of MIOX(II/III)•MI-6-one with O2. diolyl forms (48). Mössbauer spectroscopy of MIOX(II/III)•MI-6-one clearly shows that the diiron cluster is a valence-localized high-spin Fe2(II/III) cluster. Thus, the

Fe2(II/III)-ene-diolate resonance structure is the dominant contribution to the electronic structure, as was shown for the IDDs by Mössbauer spectroscopy (106).

The next step in the reaction is the addition of O2 to the MIOX(II/III)•MI-6-one cluster, for which consider two possibilities. First, O2 may add to the Fe1(II) site to

8 yield the {FeO2} complex. We label this pathway as EDD in Scheme 4-4, because addition of O2 to the Fe(II) site is similar to the reactivity of the EDDs. Perhaps more 116

importantly, this step is directly analogous to that observed in the native reaction, i.e. reaction of MIOX(II/III)•MI with O2, which results in formation of the Fe2(III/III)- superoxo complex, G. Alternatively, in analogy to the reactivity exhibited by the

IDDs, O2 may add to the C-atom of the ene-diolyl moiety with pronounced radical character (pathway labled IDD in Scheme 4) to yield a formally superoxyl-hemiketal intermediate.

The next step in both pathways is the formation of the peroxyhemiketal (PHK) intermediate, in which the peroxide moiety bridges one of the Fe sites and a C-atom of the substrate. In the EDD pathway, this would yield PHK-1, in which the peroxide is coordinated to Fe1. In the IDD pathway, the superoxyl-hemiketal complex could either attack Fe1 or Fe2 with its distal O-atom of the superoxyl moiety. Attack on Fe1 would yield the same intermediate as in the EDD pathway, PHK-1, while addition to

Fe2 would yield PHK-2. Subsequent steps in the mechanism are depicted in direct analogy those proposed for the IDDs and EDDs (86). They entail (i) heterolysis of the

O-O bond to form a Fe(III)-hydroxide species and (ii) attack of the electrons of the

C1-C6 σ-bond on the O2-derived, C-coordinated oxygen to yield the anhydride of the product, DS. Hydrolysis of the anhydride by nucleophilic attack of the Fe(III)- coordinated hydroxide on either C1 or C6 would then yield the product. Although we cannot distinguish amongst the pathways shown (and perhaps others not depicted), we prefer the path via PHK-1 for several reasons. First, in the pathway via PHK-2,

Fe1 would have no obvious role, because the mechanism would be directly analogous to that of the IDDs, which only require a Fe(III) site for catalysis. Although we

117

cannot rule out this possibility, it is important to realize that the coordination environment of the Fe(III) center in the IDDs, which involves direct coordination of two tyrosinates (86), is fundamentally different from that of Fe2 in MIOX. Second, the core geometry of the PHK-1 intermediate is identical to that of PHK proposed by

Hirao and Morokuma for the reaction cycle of the native reaction (Scheme 4-2) (26).

The observed O-incorporation pattern observed for the reaction of

18 MIOX(II/III)•MI-6-one with O2 is virtually identical to that observed for the IDDs and EDDs (98, 103). Thus, the mechanistic parallels (Criegee rearrangement followed by hydrolysis of the anhydride) are warranted. We speculate that the significant washout of the 18O label occurs on the Fe(III)-hydroxide stage prior to the nucleophilic attack on one of the carbonyl groups of the DS anhydride.

We also would like to point out that we have also considered a mechanism for the reaction of the MIOX(II/III)•MI-6-one with O2 that is directly analogous to that of the natural reaction. In this mechanism, MI-6-one would be present in its hydrated form with a gem-diol group on C6. This mechanism is inconsistent with our data, because (i) it does not account for the Δ(m/z) shift of +1 of the substrate in D2O and

(ii) the low-energy absorption features in the visible spectrum cannot be easily rationalized based on the bidentate coordination of hydrated MI-6-one via its C1 and

C6 hydroxyl groups. Both features are, however, consistent with a mechanism that invokes tautomerization of MI-6-one to its ene-diol form and its bidentate coordination.

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Conclusion

We have studied the reaction of the substrate analogue MI-6-one, in which the

C6-atom is more oxidized (by formally two oxidation units) compared to the natural substrate, MI. Although the outcome of the reactions with the MI-6-one and MI substrates appears to be directly analogous, involving the net four-electron oxidation

[cleavage of the (presumably in both cases) C1-C6 bond (a two-electron reduction) and oxidation of the C1 and C6 atoms by a total of six electrons], the mechanisms of the reactions are clearly distinct. The likely underlying cause for the alternative reactivity is the fact that MI is a redox-innocent ligand. By contrast, MI-6-one appears to be a redox-non-innocent ligand, which can tautomerize to the ene-diol form and modulate the reactivity via its oxidized ene-diolyl form. In our preferred pathway, MI-6-one binds to the Fe2(III) site as the ene-diolate in a fashion comparable to the IDDs, and O2 adds to the Fe1(II) site to generate a formally

Fe1(III)-superoxo fragment as observed in the natural reaction. This reactivity is reminiscent of the EDDs. In the natural reaction, the coordinated superoxo complex serves as the C1-H-cleaving species, while in the reaction with the MI-6-one analogue, it may directly attack the C1 center, in a step similar to the EDDs. The

PHK-1 intermediate generated in this process may be directly analogous to the proposed PHK intermediate of the natural reaction.

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Chapter 5 Conclusions and Outlook

120

Conclusions

Detailed kinetic and spectroscopic studies on MIOX have revealed that the enzyme employs an unprecedented chemical mechanism involving both substrate and O2 activation by a mixed-valent Fe2(II/III) nonheme diiron cluster (9-11). A large substrate deuterium kinetic isotope effect on decay of the intermediate, G, implies that this formally (superoxo)diiron(III/III) complex abstracts hydrogen from the substrate to initiate its oxidative ring-cleavage (10). Details of the mechanism following decay of intermediate G are currently being elucidated. H, the successor of

G, is an intermediate detected in the reaction with unlabeled substrate and the decay of H was demonstrated to be the rate-determining step in the catalytic cycle (11). The

EPR spectrum of H implies the presence of low-lying excited electronic states and reveals distinct EPR features from those of samples prepared by simply addition of

DG to MIOX(II/III). Mössbauer spectroscopy unquestionably suggests that H is a valence-localized diiron(II/III) complex, which provides valuable insight into the understanding of subsequent pathway after G, including rate-determining step and ring-cleavage. Kinetics of DG production extracted by chemical-quench-flow experiments suggest that H is either product complex or the intermediate that breaks down to product upon quenching.

The enzyme’s univalent oxidative activation of the fully reduced form by the co- substrate, O2, is an intriguing process, as it is a one-electron oxidation by the four- electron oxidant. Kinetic and spectroscopic studies clearly demonstrate that in the reaction of fully reduced MIOX(II/II) with O2, an diiron(III/III) cluster is formed as

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an intermediate and subsequently it comproportionates with extra diiron(II/II) cluster to produce the catalytic competent mixed-valent diiron(II/III) cluster. Due to the thermodynamic upper limit, a maximal 60-70% yield of MIOX(II/III) was obtained when O2/MIOX(II/II) ratio is 0.25. A mechanism is proposed to account for the stoichiometry experiment and an early formation of MIOX(II/III) which could not be attributed to the slow comproportionation.

With the use of MI analogue, MI-6-one, MIOX(II/III) can catalyze a four-electron oxidation reaction with O2 to generated a di-acid product. This dioxygenase activity of MIOX is reminiscent of key steps in the reactions catalyzed by ring-cleaving catechol dioxygenases. The tautomerization of MI-6-one from 6-keto form to ene-diol form was suggested to occur in a two-step substrate binding event. Similar to MI, binding of MI-6-one greatly perturbs the mixed-valent diiron(II/III) cluster spectroscopically. MIOX(II/III)•MI-6-one reacts with O2 rapidly to generate a stoichiometric amount of the di-acid D-saccharate (DS). The rate-limiting step of the

18 reaction is rebinding of the substrate. Isotope tracer experiments with O2 (g) indicate incorporation of two 18O atoms, of which one is partially washed out.

Although the initial steps of the reaction of the MIOX(II/III)•MI-6-one with O2 appear to be markedly different from those of the natural reaction (i.e. there is not C-

H cleavage by a superoxo-Fe2(III/III) complex), the reaction may proceed via a peroxyhemiketal intermediate that may also occur in the native reaction.

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Outlook

MIOX activates O2 and C−H with an unprecedented mixed-valent diiron(II/III) cluster and catalyzes the four-electron oxidation of myo-inositol to D-glucuronate. Its involvement of the novel O2 activation diiron cluster and a new pathway greatly expands the scope of the chemistry and increases the variety of the dinuclear non- heme-iron enzyme family. However, some challenges including the characterization of intermediates G and H, and details of the reaction steps, have still not fully understood yet. The elucidation of these remaining questions would be significantly valuable for understanding this novel and complex reaction

Characterization of Intermediate G

G accumulates to levels permitting its detailed characterization (0.4 equiv) with

D6-MI, but only to smaller level with H6-MI (less than 0.1 equiv). We can prepare samples of G for a variety of spectroscopic method in order to study its geometric and electronic structure in great detail. Although G could not accumulate to a level in which G is nearly the only species, the photo-lability of G allows us to extract the spectra of G by subtracting data of the samples enriched in G being exposed to the light from the data of parallel samples without exposure to the light. The fact that G exhibits and S =1/2 ground state will permit its detailed characterization using paramagnetic resonance methods (EPR, ENDOR, ESEEM). We expect that we can collect “orientation-selective” ENDOR spectra and obtain information about hyperfine tensors of magnetic nuclei coupled to the electronic spin. We have assigned

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G to be a formally (superoxo)diiron(III/III) cluster. If this assignment is correct, this species should exhibit anisotropic hyperfine interactions of the two 17O (I = 5/2) atoms with the electronic spin and those of the two 57Fe (I = 1/2) nuclei when using

17 57 O2 and Fe, of which the largest component is approximately 75 G. For example,

A(17O) = (76.4, 7.2, 8.2) G was observed for both O-atoms of superoxide adsorbed on

MgO, resulting in an 11-line spectrum (107). Similar values were observed for free superoxide in solution by Hoffman and co-workers (108). The spin density of the two

O-atoms of a coordinated superoxide, is likely to be different, which would result in two distinct A(17O)-tensors. Hyperfine interactions with two 17O nuclei have been observed by Hoffman and co-workers for heme-coordinated superoxo complex using

X-band EPR; the largest componednts of the A(17O)-tensors were found to be ≈ 63 G and 50 G for the two O-atoms (108). A(57Fe) tensor is also an informative value for understanding the nature of G. High-spin FeIII generally exhibits nearly isotropic A- tensors, which are dominated by the Fermi-contact term, A(57FeIII) ≈ -29 MHz (51).

Use of Substrate Analogues to Study the Mechanism of MIOX

In the introduction, we have shown the binding study of five epimers of MI by addition to MIOX(II/III) and characterization of the sample by EPR spectroscopy.

The 1- and 6(or 4)-epimers (L-chiro-inositol and epi-inositol, respectively) elicit very weak signals, whereas the 2-, 3-, and 5-epimers (scyllo-inotitol, D-chiro-inositol, and neo-inostiol, respectively) elicit stronger signals. This observation is consistent with the bidentate binding mode illustrated by crystal structure (39). The weak signals

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from the 1- and 6(or 4)- epimers could reflect either a failure of these analogues to bind or their binding in an orientation that does not elicit the EPR signal. The actual case will be diagnosed by competition experiments in which the ability of either analogue to inhibit binding of MI will be assessed by EPR. More experiments including the KD measurements of all epimers and the reactions of these complexes with O2 will provide important information for MI binding and allow for making rational choices of analogues.

In the effort for determining the substrate binding mode in the active site, the synthesis aimed to obtain a modified substrate, 6-deoxy MI was carried out by Mads-

Jacob. He synthesized a racemic mixture of the 4-deoxy and 6-deoxy analogues [(+)- and (-)-epi-quercitol]. Interestingly, precisely half of this mixture was converted by

MIOX(II/III) to a product with the molecular weight of the corresponding (3-deoxy)

D-glucuronate analogue. Kinetic studies and EPR analysis showed the turnover for 4- deoxy MI, whereas 6-deoxy MI was unable to do turnover or even bind to

MIOX(II/III), which is consistent with the deduction from crystal structure about vital functions of the hydroxyl groups on C1 and C6 of MI.

Our kinetic and spectroscopic study combined with deuterium electron-nuclear double resonance (2H-ENDOR) studies on the complex of mixed-valent MIOX(II/III)

2 with C(1-6)-[ H6]-MI by Hoffman group (21) indicated that the substrate indeed coordinate directly to the diiron(II/III) cluster. Coupling to one deuterium of the substrate is observed across the envelop of the axial EPR spectrum. We want to identify which of the six deuteria of MI is the strongly coupled deuterium by 2H-

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ENDOR experiments on MIOX(II/III)•MI complexes prepared with specifically 2H- labeled MI. C1-, and C6-2H-MI have been synthesized successfully in our group

(Denise Conner). The experiments on the complex prepared by mixing MIOX(II/III) with these specifically 2H-labeld MI are feasible. Besides the substrate binding, these experiments are important controls for ENDOR-spectroscopic characterization of intermediates G and H. In addition, we will carry out 57Fe-ENDOR experiments to further study the electronic structure of MIOX(II/III)•MI and to obtain reference spectra for ENDOR-spectroscopic characterization of G and H.

One possibly intriguing MI analogue is the L-myo-inosose-1, the C1-keto form of

MI. It was reported to be a probable intermediate in MIOX reaction (19).

Interestingly, in the mechanism proposed by Morokuma (pathway D in Scheme 1-7), a Fe-OOH C1-keto MI intermediate was assigned to be the candidate for the intermediate H (26). The subsequent steps from this species could be mimicked by the mixing of L-myo-inosose-1 with H2O2 in an electron equivalent reaction. The study of this reaction will include identifying the product of the reaction, if exhibiting turnover, defining the steady-state kinetics and indentifying any intermediate in the cycle.

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Vita Yinghui Diao

EDUCATION  Ph.D Candidate in Biochemistry and Molecular Biology, Pennsylvania State University, PA, USA, Sep, 2004- present  M.S. in Polymer Chemistry and Physics, Guangzhou Institute of Chemistry, Chinese Academy of Sciences, June 2002.  B.S. in Chemistry, Nankai University, P.R.China, June 1999.

AWARDS  R. Adams Dutcher travel award, 2008  Fundamental Subject Scholarship, Nankai University (1995, 1997, and 1998)

PUBLICATIONS 1. Bollinger, J. M. Jr., Diao, Y., Matthews, ML., Xing, G. and Krebs, C. Dalton Trans. 2009, 14(6), 905-914. 2. Xing, G., Diao, Y., Hoffart, L. M., Barr, E. W., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C. and Bollinger, J. M., Jr. Proc. Natl. Acad. Sci. U.S.A. 2006, 103(16), 6130-6135. 3. Xing, G., Barr, E. W., Diao, Y., Hoffart, L. M., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C. and Bollinger, J. M., Jr. Biochemistry, 2006, 45(17), 5402-5412. 4. Xing, G., Hoffart, L. M., Diao, Y., Prabhu, S. K., Arner, R. J., Reddy, C. C., Krebs, C. and Bollinger, J. M., Jr. Biochemistry, 2006, 45(17), 5393-5401. 5. Diao Y., Wang Q., Fu S. Letters in Applied Microbiology, 2002, 35, 451-456. 6. Diao Y., Fu S. Chemistry (Chinese), 2002, 65, w033. 7. Diao Y., Fu S.《ISGCC-2001》, 4th ISGCC, Jinan, China, 2001, May 20-24, Vol. 2:351-356