NRVS studies of the peroxide shunt intermediate in a Rieske dioxygenase and its relation to the native Fe II O 2 reaction

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1 NRVS studies of the peroxide shunt intermediate in a Rieske dioxygenase and its relation to the native Fe II O 2 reaction Kyle D. Sutherlin, a Brent S. Rivard, b Lars H. Böttger, a Lei V. Liu, a Melanie S. Rogers, b Martin Srnec, ac Kiyoung Park, ad Yoshitaka Yoda, e Shinji Kitao, f Yasuhiro Kobayashi, f Makina Saito, f Makoto Seto, f Michael Hu, g Jiyong Zhao, g John D. Lipscomb,* b Edward I. Solomon* ah a Department of Chemistry, Stanford University, Stanford, California 94305, USA. b Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA. c J. Heyrovský Institute of Physical Chemistry, The Czech Academy of Sciences, Dolejškova 2155/3, Prague 8, Czech Republic. d Department of Chemistry, KAIST, Daejeon 34141, Republic of Korea. e Japan Synchrotron Radiation Research Institute, Hyogo , Japan. f Research Reactor Institute, Kyoto University, Osaka , Japan. g Advanced Photon Source, Argonne National Laboratory, Lemont, Illinois 60439, USA. h SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA. Supporting Information Supporting Methods: ph-dependence of native O 2 reaction in BZDO Preparation of reduced BZDO BZDO in 50 mm MOPS, 100 mm NaCl, ph 6.8 was deoxygenated using a flow of argon gas at 4 C. BZDO was reduced by treating the anaerobic enzyme solution with methyl viologen (200 μm) and sodium dithionite (6.8 mm). The sample was incubated for several minutes to ensure the blue color of methyl viologen persisted. Removal of the reducing agents and transfer of reduced enzyme into 200 mm HEPES, 100 mm NaCl, 5 % glycerol ph 8.0 was accomplished using a PD-10 column (GE Healthcare) Stopped-flow analysis of a single turnover reaction Single turnover-mediated Rieske cluster re-oxidation was monitored using an Applied Photophysics SX.18MV spectrophotometer at 4 C. The instrument was rendered anaerobic by flushing with a dithionite solution. Time-based absorbance changes were measured at 464 nm. The Abs 464 of reduced BZDO (~ t = 0 s) was determined by mixing reduced enzyme with anaerobic buffer. Single turnover reactions were initiated by mixing reduced BZDO (60 μm) with benzoate (5 mm) in oxygen-gas saturated (1.8 mm) 200 mm HEPES, 100 mm NaCl, 5 % glycerol ph 8.0. Time courses were fit to multiexponential equations using Pro-Data Viewer (Applied Photophysics) as previously described. 19 Computational Details Calculation of inner-sphere reorganization energies λ i for ET was calculated using Equation S1 with DFT-derived electronic energies: λ i = (1/2)*[( ox E red ox E ox ) + ( red E ox red E red )] (S1) where subscripts denote the optimized coordinates of the reduced or oxidized state and superscripts denote the oxidation state used for the calculation. ox E red and red E ox are obtained from single-point energy calculations.to extend this for calculation of PCET, ox E red is obtained by removing the proton from the optimized reduced structure prior to performing the single-point oxidized calculation, and red E ox is S1

2 obtained by optimizing only the proton on the oxidized coordinates in the reduced state single-point calculation, following the procedure given in Ref 85. Supporting Results and Analysis PCET using the coordinated water as a proton source While we feel that it is likely that water is lost by the time that the peroxy bridge is formed, we still considered the water ligand coordinated to the Fe as a potential proton source. For the Fe III -(O 2 - )(OH 2 ) species 1 b, the PCET process generating an Fe III -(OOH)(OH) has a ΔG 0 of +3.7 kcal/mol and an λ of 2.37 ev, giving k ET = 4.2*10-7 s -1. PCET to the Fe III (OH 2 ) peroxo bridged species I 1b to generate an Fe III (OH)(OH) epoxide species has a ΔG 0 of kcal/mol and an λ of 2.35 ev, giving k ET = 540 s -1. Both rates are slower than those calculated using guanidinium as the proton source, with k ET to the superoxo being significantly slower and k ET to the peroxo bridge still faster than the rate-limiting step in single turnover. These results further support the mechanism defined in the main text, where PCET to the superoxo is slow relative to its fast reactivity with substrate, and PCET to the peroxo bridged species after this step is much more favorable and fast relative to catalysis. PCET to a 5C peroxy bridged intermediate followed by epoxide binding Removal of the water ligand from the peroxy bridged intermediate I 1b is favorable in free energy (-1.9 kcal/mol). PCET to this 5C peroxy bridged intermediate (Figure S11, left) leads to cleavage of the O-O bond and gives a 5C Fe III -OH and a benzoate epoxide (Figure S11, right), with the hydroxide proton hydrogen bonding to the epoxide. The ΔG 0 for this process is kcal/mol (using guanidinium as the proton source) and λ i is 1.12 ev, giving a k ET of 1.54*10 4 s -1 following the procedure used in Section 3.5 of the text, significantly faster than the rate-limiting step in single turnover (190 s -1 ). This intermediate is at kcal/mol relative to the Fe II -substrate starting complex (leftmost structure in Figure 6). Binding the epoxide to the 5C Fe III site enables completion of the reaction coordinate: this process is shown in Figure S12. The initial Fe-O(epoxide) distance is 4.3 Å. When the Fe-O(epoxide) distance is decreased to 3.4 Å, it becomes energetically favorable to rotate the OH proton such that it is hydrogen bonded to the O of Asn 199 rather than the epoxide O. For Fe-O(epoxide) distances shorter than 3.4 Å, structures where the hydroxy H is hydrogen bonded to O(epoxide) could not be optimized, and initial structures attempting to preserve this hydrogen bond instead converged to structures where the OH has moved such that its proton is instead hydrogen bonded with Asn199. The final epoxide-bound structure has an Fe- O(epoxide) bond length of 2.24 Å and is identical to I 3c. This coordinate, with water loss prior to PCET, is consistent with the minimal amount of 16 O incorporation observed in the single-turnover reaction of NDO with 18 O S2

3 Supplementary figures and tables Figure S1. EPR spectra of the g ~ 8 region of BZDOp and BZDO OX. Integration of the spectra in this region shows that 60% of the BZDOp sample is BZDOp itself, with 30% corresponding to BZDO OX and 10% to product-bound BZDO (labeled in blue on the figure). As BZDOp itself has no EPR intensity in this region due to its small, negative zero-field splitting (ref 16), it was quantified by integrating the initial BZDO OX signal in this region (red line) and comparing this to the integrated signal in BZDOp (blue line), with missing intensity ascribed to BZDOp, following the procedure in ref 16. S3

4 Figure S2. NRVS scans of 16 O and 18 O BZDOp. On the left is the energy range between 400 and 550 cm -1 where data were collected using 4x the measuring time used for the full scans in Figure 1. We observed potential peaks at 496 ( 18 O) and 510 cm -1 ( 16 O). To resolve these potential peaks above the noise level, we collected further data in the cm -1 energy range (marked by the black box), using 10x the measuring time. This allowed the 510 and 496 cm -1 peaks to be resolved above the noise level, as shown on the right. These data were then baseline subtracted and smoothed to generate the plot shown in the inset of Figure 1. S4

5 Figure S3. DFT structures of the side-on peroxo (Figure 2 B), side-on hydroperoxo (Figure 2 C), and endon hydroperoxo (Figure 2 D) structural candidates for BZDOp. S5

6 Figure S4. LUMO of BZDOp. Electrophilic attack would occur along the O-O vector, which the substrate cannot approach due to the positioning of Asn258. S6

7 Figure S5. DFT structures for BZDOp coordinate. Labels correspond to those given in Figure 5 in the main text. Only the lowest-energy spin state for each structure along the reaction coordinate is shown. Substrate carbons are shown in purple, protein carbons in green, oxygens in red, nitrogens in blue, iron in orange, and hydrogens in white. Second-sphere residues were included in the calculation as described in the main text but are hidden here for clarity. S7

8 Figure S6. FMOs for the Fe V (O)(OH) intermediate (I 1a ), the TS for its attack on substrate (TS 2a ), and the initial IRC product on the S = 3/2 surface (I 2a ). The most important unoccupied α (left) and β (right) FMO involved in each step of the reaction is shown, with Mulliken populations given below. The FMOs of the reactant (top) have almost no substrate π HOMO character. At the TS (middle), almost one electron (mostly α) has been transferred from the substrate π HOMO to the Fe, and at the completion of the reaction (bottom) the substrate π HOMO α and β electrons have been transferred to the Fe. S8

9 Figure S7. DFT structures for the superoxo coordinate. Labels correspond to those given in Figure 6 in the main text. Only the S = 2 structures, which are the lowest-energy spin state for each structure along the reaction coordinate, are shown. Substrate carbons are shown in purple, protein carbons in green, oxygens in red, nitrogens in blue, iron in orange, and hydrogens in white. S9

10 Figure S8. DFT structures for epoxide formation and opening coordinate. Labels correspond to those given in Figure 9 in the main text. All structures are S = 5/2, except for the S = 2 peroxo bridge I 1b,which is S = 2. Substrate carbons are shown in purple, protein carbons in green, oxygens in red, nitrogens in blue, iron in orange, and hydrogens in white. All structures include Asp303. S10

11 Figure S9. Stopped flow traces of single turnover reactions of reduced BZDO with O 2 in the presence of benzoate at ph 6.8 (black trace) and ph 8.0 (red trace) illustrating the ph independence of the reaction. Reduced BZDO (60 μm) was mixed with O 2 -saturated (1.8 mm) buffer containing benzoate (5 mm) in a stopped-flow spectrophotometer at 4 C. Solutions were prepared at ph 6.8 in 50 mm MOPS, 100 mm NaCl, or at ph 8.0 in 200 mm HEPES, 100 mm NaCl, 5 % glycerol. Product formation is complete within the time period shown. The rate constant for the product forming phase was determined by multiexponential non-linear regression fitting to be 184 ± 23 s -1 at ph 6.8 (Ref. 17) and 174 ± 10 s -1 at ph 8.0. S11

12 Figure S10. 1D linear transit for direct OH attack on the epoxide. On the left is the starting point for the transit with an O(H)-C2 distance of 2.4 Å, which is isoenergetic with I 2c, and next to it is the end point of the transit, with an O(H)-C2 distance of 1.8 Å, where the cis-diol has begun to form. On the right are the energies of structures along this coordinate, which reveal that this attack would require a barrier of at least 18.7 kcal/mol. S12

13 Figure S11. Left: 5C Fe III -peroxy-aryl radical bridged intermediate. Right: 5C Fe III -OH epoxide species generated by PCET to the structure on the left. S13

14 Figure S12. Top: Free energy of binding the epoxide to the 5C Fe III -OH species (Figure S11, right), with the OH hydrogen bonding to the hydroxide (red squares) and to Asn199 (blue triangles). Energies are quoted relative to the initial Fe III -OH epoxide unbound species (1). Bottom: Schematics of four structures along this coordinate, corresponding to the numbered points at the top. Red O atoms are the oxygens from O 2. S14

15 Table S1. NRVS vibrational parameters for BZDOp and its DFT models (all in cm -1 ). Data Side-on peroxy Side-on hydroperoxy End-on hydroperoxy νfe-o νfe-o Δ 18 O νfe-n His 284, , , , 296 ΔFe-N His Table S2. Structural parameters for the NRVS models of BZDOp (bond lengths in Å, angles in ) Side-on peroxy Side-on hydroperoxy End-on hydroperoxy O-O bond Fe-O prox bond 1.91, Fe-O H bond Fe-O-O angle Fe-His bonds 2.12, , , 2.05 S15