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Characterization of a Selenocysteine-ligated P450 Compound I Reveals Direct Link Between Electron Donation and Reactivity

Elizabeth Onderko†, Alexey Silakov†, Timothy H. Yosca‡, and Michael T. Green‡,*

‡Departments of Chemistry & Molecular Biology and Biochemistry, University of California, Irvine, CA 92697 †Department of Chemistry, Penn State University, University Park, PA 16802

Contact: [email protected]

Abstract Strong electron-donation from the axial thiolate-ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C–H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild type . incorporation was obtained using a auxotrophic E. coli strain. The intermediate was prepared with meta-chloroperbenzoic acid and characterized by UV-visible, Mössbauer, and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate-ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C–H bonds more rapidly than the wild-type intermediate.

Background Cytochrome P450s are a class of thiolate-ligated heme that are known for their ability to functionalize unactivated C–H bonds. In efforts to understand reactivity in these and other thiolate-heme systems, comparisons are often drawn between P450s and the -ligated heme . Both classes of heme share a similar active intermediate: a ferryl (or iron(IV)oxo) radical species, called compound I1-3. But, only the thiolate-ligated compound I species are known to perform C–H bond activation4-7. It has been proposed that this difference in reactivity is due to strong electron donation from the axial thiolate ligand2,8-11. However, it has proven difficult to test this hypothesis. Although a number of experiments point to this role for the thiolate2,8,10,11, a direct link between reactivity and electron donation has yet to be established in P450s. To explore the effect that electron donation has on the reactivity and electronic structure of compound I, researchers have turned to synthetic model systems and site-directed mutagenesis. Work with model systems has demonstrated that donating ligands can increase reactivity with respect to oxygen-atom transfer and C–H bond cleavage12-15. These investigations have provided considerable insight, but synthetic model systems are typically many orders of magnitude less reactive than P450s. Efforts to directly probe the role of the axial ligand via heme modifications have proven more difficult. Attempts to convert histidine-ligated heme proteins into P450-like systems have resulted in enyzmes that lack P450-style reactivity16,17. Additionally, to prevent oxidation and subsequent dissociation of the cysteine residue introduced into these systems, other distal and proximal substitutions were required, underscoring the effect that the active-site environment can have on stability as well as reactivity18,19. Efforts to introduce less electron-donating ligands such as , , and histidine into P450s have generally resulted in either apo- or misfolded protein20-22. Even when heme-containing protein was obtained, the resulting P450 variants were incapable of carrying out monooxygenase chemistry22-25. One axial ligand variant that has shown promise for the study of P450 chemistry is the substitution of selenocysteine for cysteine. The introduction of the selenolate ligand is attractive, because it represents a relatively conservative perturbation. The van der Waals radius of selenium is only 0.1 Å larger than that of sulfur26, and the formal charges of the selenolate and thiolate ligands are identical. However, the selenolate is more polarizable, has a lower redox potential, and is a better nucleophile than the thiolate27. These differences are expected to affect compound I reactivity. To date, selenocysteine has been successfully incorporated into the axial position of three different P450s28-32. Importantly, these SeP450s have been found to be catalytically competent28-30, and experiments have revealed an increase in electron donation from the axial ligand31,32. Efforts have been made to prepare compound I in these systems, but thus far only degradation products have been observed30. The analysis of P450-I reactivity with an alternative axial ligand could provide considerable insight into cytochrome P450 catalysis. Given the isoelectronic nature of the selenocysteine substitution, it would seem to be a prime candidate for these types of studies. Additionally, computations have predicted an increase in stability for the selenolate-ligated intermediate, suggesting it should be accessible via rapid mixing experiments33. Over the last few years, we have reported the preparation of compound I in several wild-type P450s1,2,34. These efforts have provided considerable insight into the conditions required for preparation of the reactive intermediate. Here, we present the capture and characterization of a selenolate- ligated cytochrome P450 compound I (Fig. 1).

Results and Discussion Selenocysteine-substituted CYP119 (SeCYP119) was overproduced using a cysteine auxotrophic E. coli strain35-37 and purified as previously described1,28,30. CYP119 possesses only one cysteine residue (the axial thiolate ligand), therefore this method resulted in selenocysteine insertion solely at the axial site. Selenocysteine incorporation at this position was determined to be ~80% by EPR analysis of ferric SeCYP119. The UV-visible and EPR spectra of SeCYP119 were found to be in good agreement with those previously reported (Supplementary Figs. 1 and 2)28-31. Upon mixing the purified SeCYP119 with meta- chloroperbenzoic acid (m-CPBA), a substantial decrease in the Soret band was accompanied by increased absorbance at 370 and 695 nm (Fig. 2a). These changes suggested compound I formation, and, indeed, through the use of target testing (TT, vida infra), a compound I-like spectrum, which we assign to SeCP119-I, could be extracted from the stopped-flow data (Fig. 2b). Maximum formation of SeCYP119-I was observed approximately 12.5 ms after mixing with one equivalent of m-CPBA, after which time the intermediate rapidly decayed to a ferric species with a Soret maximum at 406 nm (Supplementary Fig. 3). The 406 nm-absorbing SeCYP119 species was previously observed by Ortiz de Montellano and co-workers, who attributed the 406 nm absorbance to selenenic acid ligation (i.e. oxidation of the selenolate ligand)30. The selenolate-ligated compound I species was also examined by Mössbauer and EPR spectroscopies. The SeCYP119-I spectra obtained from these techniques are shown in Fig. 3. Experiments have shown that the electronic structure of compound I is best described as an S=1 ferryl unit exchange coupled to S=1/2 ligand-based radical1. The exchange coupling, J, between the spin systems generates doublet and quartet states that are mixed by the zero-field splitting, D, of the ferryl moiety to yield three Kramers doublets38. Only the lowest of these is populated at the cryogenic temperatures required for EPR and Mössbauer measurements. As a result, the spectra can be fit in either an effective S=1/2 or a spin-coupled representation (Supplementary Figs. 4-7)1. The parameters obtained from these fits are listed in Table 1. Although the Mössbauer spectrum of SeCYP119-I shows similarities to the CYP119-I spectrum1, it is clear that selenocysteine ligation has altered the heme environment. The quadrupole splitting of the selenolate-ligated system is significantly larger than the value obtained for thiolate ligated intermediates (Table 1). Additionally, variations in the isomer shift and 57Fe hyperfine couplings suggest greater electron donation from the selenolate ligand. The isomer shift decreases across the series CPO-I > CYP119-I > SeCYP119-I consistent with increasing sigma donation, while the increasingly rhombic 57Fe hyperfine couplings across this series are consistent with stronger π donation39. The effective g-values of the compound I spectrum are determined by the ratio of |J/D|38. The g-values values indicate that |J/D| is larger for SeCYP119-I than CYP119-I. Fits in the spin-coupled representation yield |J/D| = 1.4, as compared to |J/D| = 1.3 for CYP119-I. A recent study linked an increase in |J/D| to an increase in electron donation from the axial ligand, suggesting that this quantity could be a marker for compound I reactivity11. To investigate the reactivity of SeCYP119-I towards substrates, we could not use the stopped-flow techniques previously employed for the wild type intermediate: SeCYP119-I is too unstable. The intermediate cannot be prepared and subsequently mixed with substrate to obtain reaction rates, as the decay to the 406 nm species is too rapid. To obtain insight into the reactivity of SeCYP119-I, we found it necessary to generate the intermediate in the presence of substrate. This increases the rate of SeCYP119-I decay, but it also provides a window into the reactivity of SeCYP119-I that cannot be accessed with sequential mixing studies. Although quantitative reaction rates could not be determined from data sets generated in this manner, information about relative rates could be obtained, providing insight into kinetic isotope effects (KIEs) as well as the effect of the selenolate ligand on reactivity. To extract this information, we turned to TT. TT is a linear algebraic technique that uses a projection operator to determine if a given species is present in a reaction mixture 34,40. Singular Value Decomposition (SVD) of a stopped-flow data set produces abstract spectra that span the UV-visible space of the reaction mixture. As a result, the spectrum of any species in the reaction can be obtained from a linear combination of the abstract spectra. Additionally, these abstract spectra can be used to construct a projection operator. The application of this projection operator to a target spectrum (matrix × vector) reveals if the species associated with the target spectrum is in the reaction mixture. If the projection operator returns the targeted spectrum, the species is present during the reaction. If it does not, it is not. Through the application of these projection operators to the compound I spectra, we could determine if and (qualitatively) how much compound I was present in various stopped-flow data sets. Results obtained from TT are illustrated in Figure 4. The top and bottom rows show data for SeCYP119 and WT CYP119, respectively. Data sets were obtained by reacting 20 μM m-CPBA with a 20 μM/10 mM enzyme/substrate mixture (Supplementary Fig. S8). The substrate in these reactions was either perdeuterated and proteo hexanoic acid. The first column of Fig. 4 contains results for the reaction of compound I with perdeuterated hexanoic acid. It is clear that compound I accumulates in the perdeuterated reaction mixtures. In both cases, TT returns spectra that are in good agreement with the targeted compound I spectra. Comparison of the first and second columns of Fig. 4 reveals that there is a significant KIE associated with the oxidation of hexanoic acid. The application of equivalent projection operators to the proteo data sets does not return the compound I spectra. However, for the wild type reaction, TT returns a spectrum resembling that of compound I. This suggests that the intermediate is in the reaction mixture but that there is much less of it present, consistent with the more rapid oxidation of proteo substrates by compound I. In cases like this, where there is some similarity to the targeted spectrum, the incorporation of an additional abstract vector into the projection operator can often reveal the presence of the species in question. As shown in the right column of Fig. 4, the addition of another abstract vector does indeed return the compound I spectrum from the wild type data set. This is not the case for SeCYP119. The SeCYP119-I spectrum is not returned even when additional abstract vectors are added to projection operator. TT indicates that significantly less compound I accumulates in the reactions with the selenolate-ligated enzyme. To understand the implications of this result for the relative reactivities of the selenolate- and thiolate-ligated systems, the rate of compound I formation must be determined. Although formation rates in the premixed reactions were not accessible, we could measure them in the absence of substrate. Using global analysis techniques, second order rate constants for the reaction of m-CPBA with ferric enzyme were determined to be 5 × 106 M-1 s-1 and 7 × 106 M-1 s-1 for SeCYP119 and WT CYP119 (Supplementary Fig. S9), consistent with values previously reported by Ortiz de Montellano and co-workers30. Given these rate constants, the TT results indicate that SeCYP119-I is more reactive towards C–H bonds than CYP119-I: the intermediates form at essentially the same rate, but the selenolate- ligated intermediate is too reactive to be observed in reactions with the proteo substrate.

Conclusion Using insights gained from previous investigations, we have captured and characterized the selenolate-ligated compound I intermediate in CYP119. Through the application of TT to the reaction of m-CPBA with premixed protein/substrate solutions, we have examined the reactivity of the intermediate. Our results indicate that the selenolate- ligated compound I is more reactive towards C–H bonds than its wild type counterpart. A recent study has linked differences in the reactivity of P450-I and CPO-I to differences in electron donation from the axial ligand11, and it seems likely this could explain the increased reactivity of SeCYP119-I as well. The thiolate and selenolate ligands have identical formal charges, but selenium is larger, more polarizable, and a better nucleophile than . Consistent with this, the 57Fe hyperfine parameters of SeCYP119-I indicate increased electron donation from the selenolate ligand. In the first step of C–H bond activation, compound I abstracts hydrogen from the substrate to form an iron(IV)hydroxide species (compound II) and a substrate radical1,41. This process is thought to be governed, in large part, by ground state thermodynamics. The change in free energy associated with C–H bond cleavage is given by the difference between the D(C–H) bond strength of the substrate and the D(O–H) bond strength of compound II2,42. The increased reactivity of the selenolate-ligated intermediate is consistent with an increase in the compound II D(O–H). It is well known that D(O–H) is determined by the one-electron 43 reduction potential of compound I and the pKa of compound II . Given the increased electron donation from the selenolate ligand, the reduction potential of the SeCYP119-I intermediate is most likely diminished relative to that of the wild type enzyme, suggesting that the extra driving force for C–H bond activation originates from an increase in the compound II pKa that more than offsets the change in reduction potential. Efforts to determine both of these parameters are underway.

Figure 1. Selenolate-ligated compound I intermediate, a ferryl radical species.

Figure 2. The reaction of SeCYP119 with m-CPBA yields SeCYP119-I. a) 20 μM ferric SeCYP119 was reacted with 0.5, 1, 2, and 4 equivalents of m-CPBA. Spectra represent maximum formation for each reaction condition. Maximum yield was observed at 27.5, 12.5, 7.5, and 2.5 ms for 0.5, 1, 2, and 4 equivalents, respectively. b) Comparison between the spectrum of WT CYP119-I (black) and the SeCYP119-I (red) spectrum obtained from target testing.

Figure 3. CW EPR and Mössbauer spectra of SeCYP119-I a) The CW EPR spectrum of SeCYP119-I was obtained by subtracting the spectrum of WT CYP119-I from the raw data, both of which were collected at 0.63 mW and 10 K. b) The Mössbauer spectrum of SeCYP119-I was collected at 4.2 K with a 54 mT field parallel to the γ beam and the spectra of WT CYP119-I, ferric WT CYP119, and ferric SeCYP119 collected under the same conditions were subtracted from the raw data. Data are shown in black and fits are shown in blue. EPR and Mossbauer spectra of SeCYP119-I indicate an increase in the ratio between the exchange coupling parameter and zero field splitting (J/D) relative to WT CYP119-I.

Table 1. EPR and Mössbauer parameters of SeCYP119-I, WT CYP119-I, and CPO-I.

δ ΔEQ Effective S=1/2 representation Ferryl Values

(mm/s) (mm/s) gx gy gz Ax (T) Ay (T) Az (T) J/D gx gy gz Ax (T) Ay (T) Az (T) CPO-I1 0.13 0.97 1.72 1.61 2.00 -31 -30 -2 1.02 2.27 2.19 2.00 -24 -22 -2 CYP119-I1 0.11 0.92 1.96 1.86 2.00 -32 -28 -3 1.30 2.27 2.20 2.00 -23 -20 -3 SeCYP119-I 0.07 1.49 2.05 1.96 2.02 -35 -22 -5 1.43 2.29 2.22 2.02 -25 -16 -6

Uncertainties in the Mössbauer parameters listed are less than δ ± 0.02 mm/s, ΔEQ ± 0.05 mm/s, Ax,y ± 1 T, and Az ± 2 T. The line widths are 0.42 mm/s for SeCYP119-I and 0.33 mm/s for WT CYP119-I. Uncertainties in g-values are less than 0.005.

Figure 4. Target testing for the accumulation of compound I in the reaction of m-CPBA with premixed enzyme-substrate solutions. Each panel shows targeted (red) and projected (blue) spectra. If the projected and targeted spectra match, compound I accumulates in the stopped-flow reaction. SeCYP119 (top row) or WT CYP119 (bottom row) was premixed with substrate (perdeuterated hexanoic acid or hexanoic acid) and then reacted with 1 equivalent of m-CPBA (final concentrations: 6.67 μM enzyme, 6.67 μM m-CPBA, and 3.33 mM substrate). For each reaction, a projection operator was constructed from the stopped-flow data. The projection operator was then applied to the appropriate compound I spectrum. If the spectrum returned by the projection operator (shown in blue) matches the targeted compound I spectrum (shown in red), compound I accumulates in the reaction mixture. See main text for further discussion.

Methods

Materials DNA primers were purchased from Integrated DNA Technologies, Pfu polymerase was from Agilent Technologies, and the restriction enzymes, BamHI and NdeI, were obtained from New England Biolabs. L- selenocystine was purchased from Acros Organics, δ-aminolevulinic acid was from Pharmasi Chemicals Co., Ltd., IPTG and chloramphenicol were from Gold Biotechnology, ampicillin and kanamycin were from Gemini Bio Products, and 57Fe was obtained from Pennwood Chemicals, Inc. BL21(DE3) selB::kan cys51E cells were a kind gift from Dr. M. Strube. Deoxyribonuclease I from bovine pancreas, lysing enzymes from T. harzianum, m- CPBA, and hexanoic acid were from Sigma Aldrich. Hexanoic-d11 acid was obtained from C/D/N Isotopes Inc. m-CPBA was further purified by washing with pH 7.5 buffer to remove impurities. All m-CPBA stock solutions were prepared in acetonitrile and then diluted to the appropriate concentration with deionized water.

Protein Overexpression and Purification The CYP119 gene was cloned into a pET17b vector with the addition of a His6 tag using the following primers: forward (5′- CGC GGC CTC GCA TCA GGC ATA TGT ATG ACT GGT TTA G-3′) and reverse (5′- CGC GGC GTC GGA TCC TTA GTG ATG GTG ATG GTG ATG TTC ATT ACT CTT CAA CCT GAC CAC-3′). Following transformation into BL21(DE3) selB::kan cys51E, colonies were picked and patched on minimal media plates with and without cysteine. Colonies displaying the most auxotrophic behavior were selected and used to inoculate 200 mL LB cultures supplemented with 100 mg/L ampicillin, 50 mg/L kanamycin, and 60 mg/L cysteine that were grown overnight (12 h) at 250 rpm and 37 °C. 45 mL of the overnight culture were centrifuged at 5,000 × g, supernatant removed, and pellet resuspended in 10 mL of the growth media, and used to inoculate 1 L of growth media. Growth media contained per 1 L media: 1 g sodium acetate, 2 g ammonium chloride, 10 g potassium phosphate dibasic, 4.58 g sodium succinate dihydrate, and 0.8% glycerol v/v, which was supplemented with the following: 100 mL of an amino acid solution (0.4 g/L , , , glutamate, , and serine, 0.25 g/L aspartate and methionine, and 0.1 g/L , histidine, , , , , , , , , and ). Cultures were grown at 225 rpm and 37 °C until an OD of 1.2-1.3 was reached. At the appropriate OD, 0.5 mM IPTG was added. Ten minutes following the addition of IPTG, 10 mg/L chloramphenicol was added to the cultures. After an additional five minutes, cultures were centrifuged at 4200 × g for fifteen minutes. The cell pellets were washed twice with wash buffer (1 g sodium acetate, 2 g ammonium chloride, 10 g potassium phosphate dibasic, 4.58 g sodium succinate dihydrate, and 0.8% glycerol v/v per 1 L), and then resuspended in fresh growth media. After shaking at 225 rpm and 30 °C for 30 minutes, expression was induced with 0.5 mM IPTG, 0.5 mM δ-aminolevulinic, and 100 mg/L L-selenocystine. Additionally, following the final induction, the final shaking speed was reduced to 175 rpm and incubated at 28 °C for 24 hours. Cultures were then centrifuged at 7,000 × g and cell paste frozen in liquid nitrogen and stored at -80 °C until purification. Cell paste was resuspended in 50 mM potassium phosphate, 20 mM imidazole, and 500 mM NaCl buffer at pH 7.5 with the addition of 0.2 mg/mL DNase (Sigma Aldrich), and lysing enzymes from T. harzianum (Sigma Aldrich). After stirring for 30-60 minutes, the mixture was lysed using a Microfluidics M-110EH-30 microfluidizer processor. The lysate was then loaded onto a Ni-NTA agarose column, washed with 5 column volumes of lysis buffer, and eluted using 50 mM potassium phosphate, 200 mM imidazole buffer at pH 7.5. Following the removal of excess salt via stirred concentration, the protein solution was loaded onto a Q- sepharose HP column, eluted using a 0-300 mM NaCl gradient and was further purified using a Source 30-Q column. Fractions with an Rz (A417/A280) of 1.45 and higher were pooled together, exchanged into 100 mM potassium phosphate pH 7 buffer using a stirred cell concentrator with a 30 kDa filter, and used for ensuing characterizations. Selenocysteine incorporation was determined via continuous wave X-band EPR spectroscopy at 0.1 mW and 10 K. Peaks corresponding to the selenocysteine- and cysteine-ligated proteins were integrated to obtain the final incorporation percentage as described below.

Stopped Flow UV-visible Spectrophotometry Stopped flow UV-visible experiments were performed using an SFM-400 stopped flow spectrometer (Bio-Logic SA, Claix, France) with an L7893 light source (Hamamatsu, Tokyo, Japan) and a TIDAS photodiode (J&M Analytik AG, Essingen, Germany). All experiments were performed at 4 °C. Singular Value Decomposition, Global Analysis, and Target Testing were performed using Matlab.

Freeze quench Freeze-quenched samples were prepared using a freeze-quench apparatus from Update Instruments (Madison, WI). 3.5 mM ferric SeCYP119 was mixed 2:1 with 14 mM mCPBA in a 70:30 water:acetone solution and quenched at ~3.5 ms into liquid ethane (89 K). Ethane was removed under vacuum in an isopentane bath (~120 K), and the frozen sample was packed into an EPR tube and a Mössbauer cup under liquid nitrogen.

EPR Spectroscopy Continuous wave EPR spectra were obtained using a Bruker Elexys E580 X-band spectrometer with a SuperX-FT microwave bridge, an ER 4122 SHQE SuperX high sensitivity cavity, and an ER 4112-HV Oxford Instruments variable temperature helium flow cryostat. The spectrum of ferric SeCYP119 was collected at 0.1 mW and 10 K. Due to the nature of the selenocysteine incorporation, a small portion of the protein was produced with cysteine, instead of selenocysteine, as the axial ligand. The yield of the selenocysteine-substituted protein was determined using Kazan viewer44 and EasySpin45 run in a Matlab environment to simulate the spectrum of the raw ferric SeCYP119 spectrum using the simulated spectra of the thiolate-ligated (g= 2.42, 2.25, 1.92) and selenolate-ligated (g= 2.48, 2.28, 1.96) ferric enzymes. The resulting spectra were integrated and the areas used to determine the overall yield of the selenocysteine-ligated enzyme. The EPR spectrum of the SeCYP119-I freeze-quenched sample was collected at 0.63 mW and 10 K. The spectrum of the thiolate-ligated contaminating protein (WT CYP119-I sample) collected under the same conditions was subtracted to yield the spectrum shown in Figure 2a. The integral of the EPR data was fit using EasySpin 4.5.545.

Mössbauer Spectroscopy Mössbauer spectra were recorded on a spectrometer from SEE CO (formerly WEB Research) operating in constant acceleration mode in transmission geometry. The sample was kept inside an SVT-400 dewar (Janis) with a magnetic field of 54 mT applied parallel/perpendicular to the γ-beam. Isomer shifts were calibrated relative to the centroid of the spectrum of a metallic foil of α-Fe at room temperature. Data was analyzed using the program WMOSS v2.5 (WEB Research, Minneapolis, MN).

Acknowledgements This work was supported by NIH (R01-GM101390). Correspondence and requests for materials should be addressed to M.G.

Author Contributions E.O. and M.G. wrote the manuscript and designed the experiments. E.O. prepared the samples, collected stopped-flow, Mössbauer, and EPR data, and analyzed the collected data. A.S. collected and analyzed EPR data. T.Y. provided input on and assisted with experiments.

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