Formylglycine-generating binds directly at a mononuclear Cu(I) center to initiate O2 activation Mason J. Appela,b,1, Katlyn K. Meierb,1, Julien Lafrance-Vanassec,2, Hyeongtaek Limb, Chi-Lin Tsaid, Britt Hedmane, Keith O. Hodgsonb,e, John A. Tainerc,d,3, Edward I. Solomonb,e,3, and Carolyn R. Bertozzib,f,3

aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; bDepartment of Chemistry, Stanford University, Stanford, CA 94305; cMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; dDepartment of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; eStanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025; and fHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305

Edited by James E. Penner-Hahn, University of Michigan, Ann Arbor, MI, and accepted by Editorial Board Member Stephen J. Benkovic January 29, 2019 (received for review October 23, 2018)

The formylglycine-generating enzyme (FGE) is required for the Likely owing to the difficulty of isolating metallated Cu(I) protein posttranslational activation of type I sulfatases by oxidation of an crystals (13), structural characterization of an authentic Cu–FGE active-site to Cα-formylglycine. FGE has emerged as an complex has remained elusive. Additionally, the manner in which enabling biotechnology tool due to the robust utility of the alde- the monocopper site reacts with O2, and the reactive intermedi- hyde product as a bioconjugation handle in recombinant proteins. ates responsible for substrate oxidation are unclear. Therefore,

Here, we show that Cu(I)–FGE is functional in O2 activation and elucidation of the catalytic mechanism of FGE is fundamental to a reveal a high-resolution X-ray crystal structure of FGE in complex broader understanding of copper-mediated biotransformations. with its catalytic copper . We establish that the copper Here, we present the X-ray crystal structure of Cu-bound FGE atom is coordinated by two active-site cysteine residues in a nearly at a resolution of 2.2 Å. The linear cuprous dithiolate ligand linear geometry, supporting and extending prior biochemical and system observed in the holoenzyme structure is unusual for a CHEMISTRY structural data. The active cuprous FGE complex was interrogated copper oxidase and is superficially similar to well-characterized directly by X-ray absorption spectroscopy. These data unambigu- Cu(I) transporters and chaperones (14). Additionally, we report ously establish the configuration of the resting enzyme cen- the electronic absorption and electron paramagnetic resonance ter and, importantly, reveal the formation of a three-coordinate (EPR) features of a transient cupric-FGE complex, providing tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calcula- Significance tions. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a Many harness the energy of molecular oxygen to cata- BIOCHEMISTRY detailed mechanistic framework for understanding why nature lyze diverse transformations. However, reaction with oxygen is chose this structurally unique monocopper to catalyze kinetically challenging and must be precisely controlled to prevent oxidase chemistry for sulfatase activation. cellular damage. The formylglycine-generating enzyme activates its targets by utilizing a mononuclear copper center to facilitate formylglycine | copper oxidase | metalloenzyme | X-ray spectroscopy | oxidation of a substrate cysteine. We reveal the structure of its bioinorganic chemistry atypical copper active site and discover that substrate binding directly to copper precedes and initiates reactivity toward oxygen. he formylglycine-generating enzyme (FGE) catalyzes the We furthermore identify a transient intermediate that may pro- Tcotranslational or posttranslational activation of type I sul- vide evidence for the involvement of an elusive catalytic species. fatases in eukaryotes and aerobic microbes (1). This is accom- This work uncovers a distinct strategy among copper enzymes plished by oxidation of a sulfatase active-site cysteine residue that exploits a dynamic to tightly couple the activa- located in the minimum consensus sequence CXPXR to Cα- tion of oxygen with selective substrate oxidation. formylglycine (fGly), a critical cofactor for the hydrolysis of sulfate ester substrates (Fig. 1). Dysfunction of FGE in humans Author contributions: M.J.A., K.K.M., J.L.-V., J.A.T., E.I.S., and C.R.B. designed research; results in a congenital disease, multiple sulfatase deficiency, M.J.A., K.K.M., J.L.-V., and H.L. performed research; M.J.A., K.K.M., J.L.-V., H.L., C.-L.T., B.H., K.O.H., J.A.T., E.I.S., and C.R.B. analyzed data; and M.J.A., K.K.M., J.A.T., E.I.S., and which originates from a global decrease of sulfatase function (2, C.R.B. wrote the paper. 3). The promiscuity of FGE has enabled its use in biotechnology Conflict of interest statement: C.R.B. is a cofounder and member of the Scientific Advisory and therapeutic applications, such as site-specific drug attach- Board of Redwood Bioscience (a subsidiary of Catalent, Inc.), which has exclusive rights to ment to fGly in monoclonal antibodies (4–6). the SMARTag technology based on protein modification by FGE. C.R.B. is also a cofounder Initially, FGE was thought to catalyze Cys-to-fGly conversion of Palleon Pharmaceuticals, Enable Biosciences, and InterVenn Biosciences, and a member using an unprecedented cofactorless oxidase/monooxygenase of the Board of Directors of Eli Lilly & Co. mechanism, relying on an essential redox-active cysteine pair (7). This article is a PNAS Direct Submission. J.E.P.-H. is a guest editor invited by the However, in 2015, Holder et al. (8) provided evidence for acti- Editorial Board. vation and stoichiometric Cu binding in Streptomyces coelicolor Published under the PNAS license. and human FGEs, implicating FGE as a Cu-dependent metal- Data deposition: The X-ray crystallography data have been deposited in the Protein Data loenzyme. Further investigation was carried out by Knop et al. Bank, www.wwpdb.org (PDB ID code 6MUJ). − 17 1M.J.A. and K.K.M. contributed equally to this work. (9), detailing that high-affinity (Kd ∼10 M) copper binding is dependent on the aforementioned active-site cysteine pair (10). 2Present address: Protein Chemistry, Genentech, South San Francisco, CA 94080. This was unexpected because typical mononuclear copper oxi- 3To whom correspondence may be addressed. Email: [email protected], edward. dases contain N- and O-ligands at higher coordination states [email protected], or [email protected]. (11). FGE crystal structures bound to catalytically inactive Ag(I) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and Cd(II) have provided structural evidence for a dicysteine 1073/pnas.1818274116/-/DCSupplemental. binding motif reminiscent of known Cu(I) binding proteins (12).

www.pnas.org/cgi/doi/10.1073/pnas.1818274116 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 HS O H treatment (SI Appendix, Fig. S4). Thus, reducing equivalents for this process ultimately originate from FGE cysteine . Binding of Cu(I) to scFGE was also observed; anaerobic addition FGE, O + − 2 of one equivalent of Cu(MeCN)4 PF6 yielded stable holoenzyme N N with >0.98:1 Cu:FGE after buffer exchange. H H O O Single-Turnover Activity of Cu(I)– and Cu(II)–FGE. The catalytic Cys fGly properties of FGE in each copper oxidation state have not been addressed directly in prior studies. Although the activity of

Fig. 1. The O2-dependent conversion of cysteine to Cα-formylglycine (fGly) Cu(I)-loaded FGE has been demonstrated under steady-state catalyzed by FGE. conditions (9, 10), our data prove that apo-FGE binds both Cu(I) and Cu(II). The observed rate constant for the autor- − − eductive decay of Cu(II)–scFGE (8 × 10 4 s 1) is more than two −1 direct evidence that FGE can bind both copper redox states with orders of magnitude lower than that of kcat for scFGE (0.3 s ) the same active-site ligands. By directly interrogating Cu(I)–FGE (8). Therefore, it is kinetically feasible that Cu(II)–scFGE could using X-ray absorption spectroscopy (XAS), the linear two- be catalytically competent in the presence of substrate and O2. coordinate crystallographic model was confirmed in solution. As the oxidation state of the resting enzyme places specific Moreover, these studies reveal a shift to a three-coordinate constraints on the mechanistic pathway available for substrate Tris(thiolate) Cu(I) complex upon inner-sphere substrate oxidation, explicitly testing the catalytic ability of each Cu–FGE binding, before oxygen binding and activation. Finally, in the redox state is of fundamental importance. reaction of the preformed enzyme–substrate complex with O2,a Single-turnover reactions containing 1:1 scFGE:substrate were transient intermediate is observed by stopped-flow absorbance employed to compare the activity of Cu(I)– and Cu(II)–scFGE spectroscopy. Together, these results represent the structural during the first turnover (Fig. 4). These assays used a 15-mer characterization of Cu-bound FGE in both redox states, and peptide substrate (DNP-scP15) containing a 13-residue recog- definitively identify the O2 activating site. nition motif from an S. coelicolor sulfatase and an N-terminal chromophore. To account for the inherent instability of the Results Cu(II)–scFGE complex, one equivalent of Cu(NO3)2 was added Discovery and Characterization of a Transient Cu(II)–FGE Complex. to apo-scFGE immediately before each reaction time course. Initial interrogation of the S. coelicolor FGE (scFGE) copper Within 2 min, Cu(I)–scFGE achieves nearly 70% conversion of center was pursued using UV-vis absorption and EPR (Figs. 2 and substrate to fGly, compared with 10% conversion by Cu(II)– 3), revealing characteristic features of an explicit Cu(II)–FGE scFGE. When the reaction was preceded by a 5-min treatment complex. Aerobic mixing of Cu(II) solution with apo-scFGE with excess DTT, Cu(II)–scFGE was activated approximately resulted in a vivid blue solution. This color change is associated eightfold. Unlike during multiturnover conditions (9), DTT does with at least two absorbance features with maxima at 348 and not further activate Cu(I)–scFGE for a single turnover, sug- 375 nm that saturate in intensity at ∼1:1 Cu(II):FGE (e = gesting that the input of external electrons is not required to − − 375 4,940 M 1·cm 1; SI Appendix,Fig.S1). Curiously, these solu- resolve an intermediate and instead occurs after product for- tions turned colorless after ∼10 min. Stopped-flow UV-vis ab- mation. These data conclusively identify Cu(I)–scFGE as the sorption spectroscopy was employed to measure the kinetics of catalytic resting enzyme. the chromophoric species formed by mixing 1:1 apo-scFGE and X-Ray Crystal Structure of the Cu–scFGE Holoenzyme. Cu(NO3)2 solution anaerobically. Absorbance features in the With the benefit 320- to 450-nm region form immediately and maximize in in- of an apo X-ray crystal structure (15), scFGE was likewise chosen tensity within the first 175 s, then decay to baseline over 17 min for structural studies of the reconstituted holoenzyme. Using the (Fig. 2A). The observed rate constants for formation and decay copper activation procedure described previously (8), a crystalliza- of the 375-nm species at 100 μM (apparent) Cu(II)–scFGE tion screen was performed around the conditions used to generate −2 −1 −4 −1 were k1 = 2.45 × 10 s ,andk2 = 8 × 10 s , respectively. the apo enzyme crystals (15). Initial screens with premetallated These features are absent in the inactive C272A/C277A double enzyme and with supplemented copper salts failed to yield copper- mutant (SI Appendix,Fig.S2), providing evidence for the for- containing crystals. This suggested that copper may be released mation of a transient Cu(II)–Cys(272,277) complex. However, during crystallization, or that a disulfide active-site conformation the presence of additional ligands from solvent or protein is not that excluded copper entirely was a prerequisite for crystallization. discernible from these data. Successful crystallization of the scFGE C277A mutant excludes the The instability of the Cu(II)–Cys(272,277) complex can result − from copper dissociation or 1e reduction to form a transparent Cu(I)–FGE complex, among other mechanisms. To evaluate the 0.12 dissociation pathway, the fraction of FGE-bound copper fol- AB350 nm lowing anaerobic reconstitution was monitored as a function of 0.16 375 nm 565 nm time (Fig. 3A). Compared with the complete loss of absorption 0.12 0.08 bands (Fig. 2A), the Cu:FGE ratio only declined from 0.99 to bance (a.u.) rbance (a.u.) – 0.08 r 0.90. Therefore, the disappearance of the chromophoric Cu(II) 0.04 Abso Cys(272,277) complex cannot be attributed to copper release. To 0.04 Abso assess whether a reductive pathway is instead responsible, X- 0 0 band EPR spectra were collected in a parallel time course. 300 400 500 600 700 0 200 400 600 800 1000 The EPR spectra (Fig. 3B) contain a mixture of at least three Wavelength (nm) Time (s) species. Two species having similar g values and hyperfine cou- Fig. 2. Addition of Cu(II) to FGE forms a copper complex distinguished by pling constants are already formed by 86-s post-Cu(II) addition transient UV-vis absorbance features. (A) Stopped-flow UV-vis spectra of apo (black), and nearly disappear over 1 h (purple). A third intense – = scFGE following mixing with one equivalent of Cu(NO3)2. Features of the Cu(II) feature near g 2.00 may be tentatively assigned as an organic scFGE complex grow from 1 s (black) to a maximum at 175 s (blue), and decay radical. The loss of EPR-active copper occurs with comparable from 175 to 1,000 s (red). (B) The baseline-subtracted kinetic traces of the 350-, kinetics to the decay of Cu(II)–scFGE absorbance features and is 375-, and 565-nm absorbance features. The kinetic trace of the 375-nm feature − − consistent with the autoreduction of Cu(II)–scFGE to Cu(I)– was fit to a double-exponential equation, Y = A(1 − e k1t) + Ae k2t + C, using −2 −4 −1 scFGE. Nonreducing SDS-PAGE demonstrates concomitant nonlinear regression, yielding rate constants k1 = 2.45 × 10 ± 1 × 10 s for −4 −6 −1 disulfide-mediated oligomerization of scFGE following Cu(II) growth and k2 = 8 × 10 ± 2.4 × 10 s for decay. Errors are the SEs of the fit.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1818274116 Appel et al. Downloaded by guest on September 25, 2021 AB thiolate ligand of C277 (Fig. 5A). Although the redox state of copper in crystallo was not explicitly known, Cu(I) is inferred 1.0 from the two-coordinate linear ligand geometry, and the propensity 0.8 for copper photoreduction during X-ray diffraction data collection

"/dH at 77 K (21). This crystal structure is particularly revelatory because X 0.6 d this binding motif is not present in previously described copper fraction of l oxidases/oxygenases (11), and must confer reactivity that is uniquely

mo bound copper 0.4 suited to catalysis of O2-dependent fGly formation. 0.2 XAS and Extended X-ray Absorption Fine Structure of Cu(I)–scFGE. 0 0 1020304050602600 2800 3000 3200 3400 3600 3800 Our biochemical data and the detailed atomic structure pro- time following Cu(II) addition (min) B(G) vided by X-ray crystallography define the importance of the – – – Cu(I) scFGE complex. XAS (22) was used to determine the Fig. 3. The Cu(II) FGE complex undergoes autoreduction to Cu(I) FGE. (A) coordination number, ligand identity, and bond distances of The fraction of FGE-bound Cu remaining after anaerobic addition of one Cu(I)–scFGE in solution. The Cu(I)–scFGE holoenzyme was equivalent of Cu(NO3)2 to apo FGE and incubation at 25 °C, as determined by biquinoline assay of buffer-exchanged aliquots at each time point. Error bars prepared anaerobically and found to be stable at a concentration of represent the SD of three replicates. (B) 77 K, X-band EPR quantification of 1mMin50mMTris,pH9.0,0.5MNaCl,and10%glycerol.The – total Cu(II) following addition of Cu(NO3)2 to apo scFGE in a parallel ex- Cu K-edge XAS spectrum of a frozen solution of Cu(I) scFGE periment to A, but without buffer exchange following incubation. Each (Fig. 6A, black) revealed an intense absorption feature in the spectrum represents a single time point of an anaerobic solution of Cu(II)– 8,984-eV region, with a normalized intensity near 1.0, correspond- scFGE: 90 s, black; 490 s, red; 1,260 s, blue; 3,470 s, purple. ing to the Cu(I) (1s → 4p) transition for a linear two-coordinate complex (22, 23). Extended X-ray absorption fine structure (EXAFS) spectra latter model. Instead, an irreversible copper dissociation mechanism were collected to identify the ligand environment at copper likely accounts for the absence of bound copper among crystalli- (24). EXAFS data and its Fourier transform are shown in Fig. zation trials. Following copper binding, the extended period of 6B (black). Using density functional theory (DFT)-optimized crystal growth may allow copper to dissociate via oxidation of the parameters as a starting point, these EXAFS data were used to putative cysteine ligands, yielding apo crystals. The apparent in- extract information about bond lengths and ligand identities. stability of Cu–FGE may be related to the autoreduction reaction The best-fit model of the Cu(I)–scFGE EXAFS data included CHEMISTRY pathway of Cu(II)–scFGE and is consistent with previous bio- two ligands at an average Cu–S bond distance of 2.14 Å, chemical findings (10). in good agreement with the crystal structure (Table 2). Full With this in mind, a multisolution crystal soaking procedure EXAFS fitting results are shown in SI Appendix,Fig.S6.In was employed. In brief, crystals were harvested, reduced with addition to collecting and analyzing XAS and EXAFS spectra DTT, soaked with CuCl2, and last, soaked in cryoprotectant for of Cu(I)–scFGE, we also characterized the fully autoreduced storage in liquid N2. This sequence resulted in a reconstituted enzyme. Samples of autoreduced scFGE were prepared an- holoenzyme crystal structure of FGE with copper, its native and aerobically by allowing Cu(II)–scFGE to decay to the colorless – ∼ sole functional metal cofactor (Fig. 5). Holoenzyme crystals Cu(I) scFGE complex ( 1 h). Comparison of the Cu K-edge BIOCHEMISTRY diffracted to a resolution of 2.2 Å, and phases were determined XAS data for both complexes revealed that autoreduction re- by molecular replacement using apo-scFGE (PDB ID 2Q17). sults in an identical cuprous complex to that formed via direct The resulting structure contains five per asymmetric Cu(I) binding to scFGE (Fig. 6 A and B,blue). unit with RMSD ∼0.21–0.27 Å between molecules. X-ray data collection and refinement statistics support the accuracy of the XAS and EXAFS of Substrate-Bound Cu(I)–FGE. Previous experi- refined structure (Table 1). The well-conserved structure in all mental and computational studies using Cu-free apo-FGE have crystallographically independent molecules improves confidence in identified a shallow binding pocket for peptide substrates that the active-site structure and geometry for providing mechanistic may orient the substrate cysteine residue toward C272 and C277 understanding, as seen for the dioxygen reactive sites in (25). However, the interaction of substrate with the copper dismutases (16–18). From the crystal packing, a dimeric form (chain center has remained ambiguous. XAS was utilized to test AB or DE or C2) of Cu(I)–scFGE was implicated as a stable as- whether the substrate thiolate binds merely in the vicinity of the 2 sembly by PISA analysis (19) (surface area, 21,390 Å ; buried area, copper center or ligates it directly (12). Two factors were of 2 assembly diss 5,160 Å ; ΔG = −115.2 kcal/mol; ΔG = 5.1 kcal/mol). concern in evaluating substrate binding. First, that Cu(I)–scFGE However, size-exclusion chromatography coupled multiangle light and its peptide substrate will form an inert complex under an- scattering demonstrates that both apo-scFGE, and Cu(I)–scFGE aerobic conditions and, second, that substrate binds with suffi- with peptide substrate, are monomers in solution (SI Appendix, cient affinity to the Cu(I)–scFGE complex to achieve sample Fig. S5A). concentrations required for XAS studies. Each contains a Cu , identified by a strong anomalous peak in the experimental electron density map, that is bound to two cysteine residues (C272, C277) and a Ca atom bound to D208 and N197 (Fig. 5A). The coordination environ- 100 ment of the Cu center features a linear, two-coordinate metal- 75 binding site composed of both active-site , is iso- – 50 Cu(I)-FGE structural to that of the inactive Ag(I) FGE (PDB ID 5NXL; Cu(I)-FGE + DTT

RMSD, 0.61 Å), and is superficially similar to active sites of (µM) [fGly] – – 25 Cu(II)-FGE cuprous trafficking proteins (Fig. 5A) (12, 20). The S1 [Cu] S2 angle Cu(II)-FGE + DTT is 171°, and S–Cu bond distances are between 2.1 and 2.2 Å for 0 C272 and C277 among five molecules. The apo and holoenzyme 020406080100120140 structures align closely with RMSD ∼0.38 Å (Fig. 5B), suggesting a Time (s) largely preformed Cu atom binding site in the apo enzyme. Fig. 4. Single-turnover activity assays of Cu(I)- and Cu(II)-loaded scFGE. Re- Notably, a bond between the side chains of the actions contained 100 μMFGEand100μM DNP-scP15 peptide substrate at conserved (SI Appendix, Fig. S5B) D47 and R279 residues is 4 °C, in the presence or absence of 2 mM DTT. The progress curves were fit to a −kobst specific to the holoenzyme and appears to organize an in- single-exponential equation, Y = Ao(1 − e ), using nonlinear regression teraction network that may be important for positioning of the (solid and dashed lines).

Appel et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 25, 2021 Fig. 5. X-ray crystal structure of copper-bound scFGE. (A) Cu bound at the FGE active site, coordinated by C272, C277, shown with adjacent active-site residues. The electron density map is overlaid as gray mesh (2Fo-Fc, 1 σ), and the anomalous difference map as purple mesh (4 σ). Cu and Ca are displayed as brown and green spheres, respectively. (B) Alignment of the Cu–scFGE holoenzyme crystal structure (cyan) to the apo enzyme (orange; PDB ID 2Q17) shown for the active-site region, with RMSD ∼0.38 Å.

A 14-mer substrate (scP14) composed of 13 residues from an bound crystal structure (PDB ID 5NXL) (12). Single-turnover S. coelicolor sulfatase sequence (National Center for Bio- activity of Cu(I)–FGE supports its assignment as the catalytic re- technology Information: WP_011031740.1), and a C-terminal dox form and corroborates the relevance of our structural data. We tyrosine was selected for these studies. The KM of scP14 was also report the discovery and characterization of a noncatalytic, or found to be >10-fold lower (6.0 ± 0.8 μM; SI Appendix, Fig. S7) perhaps precatalytic, Cu(II)–scFGE complex. The kinetics of Cu than that used in a prior study (8). Therefore, we expected that (II)-dependent UV-vis and EPR features demonstrate the in- this feature may increase the equilibrium fraction of the Cu(I)– stability of this complex, which decays to a species that is spectro- scFGE:scP14 complex for high-quality XAS samples. scopically and catalytically equivalent to Cu(I)–scFGE. These data The Cu(I)–scFGE:scP14 complex was isolated anaerobically provide a coherent model for an autoreduction process that main- by the addition of one equivalent peptide to enzyme at a final tains bound metal, but converts Cu(II)–scFGE to a stable Cu(I) concentration of 1 mM each. The Cu K-edge XAS spectrum of conformation capable of catalysis. It is unclear whether the ligand the Cu(I)–scFGE:scP14 complex revealed a decrease in the in- environment of transient Cu(II)–scFGE is analogous to that of the tensity of the 8,983- to 8,985-eV feature compared with that of inactive Cd(II)–FGE structure (PDB ID 5NYY) (12). Cu(I)–scFGE (0.77 vs. 1.0). An intensity decrease of this mag- FGE is functionally distinct from, and phylogenetically un- nitude is indicative of a change from two- to three-coordinate, related to, known Cu(I) binding proteins (14, 20, 26). For ex- for a sizable fraction of the enzyme (Fig. 6C) (23). The increase ample, the Cu-responsive transcriptional activator (CueR; PDB in the coordination number is also supported by the corre- ID 1Q05) also binds copper in a linear bis(thiolate) geometry, – 4 −21 sponding EXAFS data, which were best fit with three Cu S but with 10 -fold greater affinity (Kd ∼10 M) than FGE (27) bonds at 2.22 Å (Table 2, Fit 3B). The fit with two short and one longer Cu–S was attempted but excluded because of the negative 2 σ value (Table 2, Fit 3C). Replacement of one Cu–S path by a Table 1. Crystallography data collection and Cu–O path was also tested but led to a larger error and very high refinement statistics σ2 value for the Cu–O path (Table 2, Fit 3D). These data provide – experimental verification of substrate binding directly to the Cu(I)- Parameters Cu scFGE scFGE active site. Data collection Space group P3 21 O Activation at the FGE Copper Center Reveals a Catalytic Intermediate. 1 2 Cell dimensions To interrogate Cu–FGE during catalysis, stopped-flow UV-vis ab- a = b, c, Å 140.0, 217.1 sorption spectroscopy was used to monitor changes in absorption α = β γ spectra recorded from 5 ms to 20 min following mixing of Cu(I)– , (°) 90, 120 Resolution, Å 37.89–2.25 (2.33–2.25) scFGE:scP14 (anaerobic) with O2-saturated buffer. The time- dependent absorption spectra of this reaction revealed a transient Redundancy 10.8 (8.8) feature at 425 nm (Fig. 7, blue). Completeness (%) 93.95 (83.18) Mean I/sigma (I) 12.72 (0.65) However, mixing Cu(I)–scFGE (anaerobic) alone with O2- saturated buffer did not produce this feature (Fig. 7, black). CC1/2 0.998 (0.538) Interestingly, the reaction of autoreduced Cu(II)–scFGE:scP14 Refinement (anaerobic) with O2-saturated buffer yielded an identical chro- No. reflections 110,236 mophoric species that accumulated to approximately the same Rwork/Rfree 0.2024/0.2456 amounts with the same time dependence as those observed for No. atoms the Cu(I)-loaded scFGE:scP14 (Fig. 7, red). This result confirms Protein 11,267 that when Cu(II)–scFGE is allowed to completely autoreduce, it Ligand/ion 80 converts to a form that is also catalytically competent. These 653 observations identify the 425-nm species as the key intermediate Average B-factor 48.58 observed during FGE catalysis, and its characterization is cur- Protein 48.54 rently underway in our laboratories. Ligand/ion 64.87 Water 47.19 Discussion RMSDs Here, we present the X-ray crystal structure of the FGE holo- Bond lengths, Å 0.008 enzyme containing its native copper cofactor, revealing a Cu Bond angles, ° 0.95 binding site that shows a surprising linear coordination with two PDB code 6MUJ cysteine ligands, and is validated in solution by XAS/EXAFS. Our Cu(I)–scFGE active site is in good agreement with the Ag(I)- Statistics for the highest-resolution shell are shown in parentheses.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1818274116 Appel et al. Downloaded by guest on September 25, 2021 1.2 The prediction that the substrate thiolate binds directly at A B 4

n copper in the Michaelis complex is verified experimentally by a 1.2 2 tio

p decrease in intensity of the 8,984-eV XAS peak and a markedly 0.8 0

sor – x EXAFS different EXAFS beat pattern relative to Cu(I) scFGE (Fig. 3 b 0.8 -2 k -4 6D). The change in coordination number upon substrate binding 0.4 24681012 – ∼ FT Intensity is correlated with an elongation of the Cu S bonds from 2.14 Å 0.4 k (Å-1) in Cu(I)–scFGE to ∼2.22 Å in Cu(I)–scFGE:scP14. The Cu– Normalized A 0 0 scFGE crystal structure, combined with enhanced information 8970 8980 8990 9000 9010 9020 0123456 from XAS, forms the basis of more detailed electronic structure Energy (eV) R(Å) calculations of the Cu(I)–FGE and Cu(I)–scFGE:scP14 copper C1.2 D 4 centers. DFT geometry optimization of crude scFGE:substrate 1.2 2 y docking models show that bond elongation is accompanied by a 0.8 0 change in S–Cu–S bond angle from 172° in resting Cu(I)–scFGE x EXAFS 0.8 3 -2 – k to 134° in Cu(I) scFGE:substrate. Importantly, these structures -4 can be used to provide valuable insight regarding changes to the 0.4 2 4 6 8 10 12 FT0.4 Intensit k (Å-1) electronic structure of the copper active site, which in turn alter the O2 reactivity of the scFGE complex. Normalized Absorption – 0 0 Stopped-flow experiments show Cu(I) scFGE is relatively 8970 8980 8990 9000 9010 9020 0123456 stable under both aerobic and anaerobic conditions. However, Energy (eV) R(Å) upon substrate binding, an O2-activation reaction is available to Fig. 6. XAS of the Cu(I)–scFGE, Cu(II)–scFGE → Cu(I)–scFGE (autoreduced), generate an intermediate. To our knowledge, no other mono- and Cu(I)–scFGE:scP14 complexes. (A) Cu K-edge XAS spectra, and (B) EXAFS nuclear copper enzyme is known to bind substrate and a reactive data (Inset) and non–phase-shift-corrected Fourier transforms for Cu(I)–FGE O intermediate simultaneously at the metal center. For com- 2 – (black) and Cu(II)–scFGE → Cu(I)–scFGE (autoreduced) (blue). (C) Cu K-edge parison, galactose oxidase catalyzes the 2e oxidation of D-ga- XAS spectra, and (D) EXAFS data (Inset) and non–phase-shift-corrected lactose to D-galacto-hexodialdose directly from a Cu(II)/cofactor Fourier transforms for Cu(I)–FGE (black) and Cu(I)–FGE:scP14 (red). These radical pair through an inner-sphere complex with the substrate data are representative of at least three replicates. hydroxyl, and only then binds and reacts with O2 to regenerate

the oxidized resting state (30). Thus, the unique susceptibility of CHEMISTRY FGE’s substrate to nonproductive oxidation appears to – – – – φ and with a disparate Cβ1 S1 [Cu] S2 Cβ2 torsion angle ( )of necessitate a mechanism that tightly controls the location of both φ = 19° (FGE) vs. φ = 108° (CueR). The unique deviation of φ in activated oxygen species and the substrate sulfur atom during FGE compared with CueR and other Cu(I) binding proteins (20) turnover. may be viewed as a structural indication that its copper site has A mechanistic proposal consistent with the results of this study evolved to perform the dynamic requirements of substrate binding (Fig. 8) supports and extends predictions made by Seebeck and and oxidase/oxygenase catalysis. The functional divergence of coworkers (10, 12), with evidence for (1) and (2) presented here. these two copper sites is likely related to structural differences. Substrate binds to the two-coordinate Cu(I) active site via the The FGE Cu center is surrounded by polar residues and is ac- cysteine sulfur to yield a three-coordinate Cu(I)–scFGE:sub- BIOCHEMISTRY cessible to substrate, solvent, and O2 (SI Appendix, Fig. S8A). strate complex that is primed for reaction with O2. H-atom ab- CueR forms a buried Cu(I) site: It is closed by a shift at the dimer straction (HAA) of the pro-(R)-β-hydrogen from the substrate cysteine is the rate-determining step (k /k = 3.0–3.7) (12) and interface of three helices (28) and is protected from solvent and H D – O binding by hydrophobic residues (SI Appendix, Fig. S8B). Al- involves an activated oxygen species, putatively a Cu(II)–OO· 2 3 – though the O2 reactivity of CueR has not been tested directly, the ( ). HAA by a superoxo intermediate would generate a Cu(II) role of CueR under aerobic copper stress in Escherichia coli sug- OOH species with a carbon-centered radical on substrate (4a) gests it is inert (29). or, depending of the rate of product release relative to that of C=S bond formation, species (4b). In the latter, HAA from the substrate results in the rapid reduction of Cu(II) to Cu(I) and the Table 2. EXAFS fitting results concomitant formation of a thioaldehyde weakly bound to

† 2 2‡ § the metal via the substrate sulfur lone pair. Indeed, this process Complex Fit CN/path* R,Å σ ,Å ΔE0, eV Error F

Cu(I)–FGE 1A 1 Cu-S 2.14 67 −13.68 0.41 − 1B 2 Cu-S 2.14 390 13.79 0.29 0.02 1C 3 Cu-S 2.14 641 −14.14 0.34 – − Cu(I) scFGE 2A 1 Cu-S 2.13 97 15.75 0.43 0.015 (autoreduced) 2B 2 Cu-S 2.13 420 −15.60 0.30

2C 3 Cu-S 2.13 669 −15.82 0.34 (a.u.) Cu(I)–FGE:scP14 3A 2 Cu-S 2.23 365 −9.79 0.36 0.01 3B 3 Cu-S 2.22 583 −10.61 0.30 − 0.005 3C 2 Cu-S 2.17 265 13.01 0.28 425 nm

1 Cu-S 2.28 −64 A 3D 2 Cu-S 2.22 365 −10.78 0.35 0 1 Cu-O 2.57 1,284 024681012 3E 4 Cu-S 2.22 775 −10.96 0.33 Time (s)

Errors in coordination numbers are ±25%, and those in the identity of the Fig. 7. Observation of an intermediate during FGE catalysis. Stopped-flow scatterer Z are ±1. The best acceptable fit for each complex is shown in bold UV-vis spectra of a transient absorbance feature at 425 nm that is dependent

font. on the presence of Cu holoenzyme, substrate, and O2. Kinetic traces at *CN is coordination number. 425 nm are shown for the following: anaerobic Cu(I)–scFGE mixed with O2- † The estimated SDs in R are ±0.02 Å. saturated buffer (black), anaerobic preformed Cu(I)–scFGE:scP14 complex ‡ σ2 5 – The values are multipliedP by 10 . P mixed with O2-saturated buffer (blue), and anaerobic autoreduced Cu(I) § 6 6 2 1/2 The error F is given by [ k (χexptl − χcalcd)(2)/ k χexptl ] . scFGE:scP14 with O2-saturated buffer (red).

Appel et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 25, 2021 1 2 3 reduce O2 to H2O. It has been proposed that the Cu(I)–OOH (5) Cys Cys Cys277 S 277 S 277 S O O intermediate is resolved by transfer of an oxidative equivalent to S H O + Cu+ S H 2 Cu2+ ’ – Cu H H one of the enzyme s sulfur ligands; the formation of an S Oin- H S R S H S S termediate would accompany a change in the ligand occupancy at Cys272 Cys272 R Cys272 R Cu(I), and reduction from an external thiol source would follow (12). This proposal may be verified experimentally by chemical H2O2 or 2 H2O + trapping, or spectroscopically using the EXAFS approach in this 1H or (2e + 2H+) 4a – Cys277 S O OH report. The direct 2e reduction of Cu(I)–OOH offers an alternative 5 Cu2+ pathway but would rely on a sufficiently high Cu H · • to forestall detrimental formation of Cu(II)/ OH. Cys277 S S S S Cys272 A mechanistic understanding of FGE catalysis has direct sig- + R Cu O OH nificance to the understanding of human sulfatase biology and R H S Cys272 4b diseases (35), and can be exploited to facilitate the production of H2O Cys277 S O OH therapeutics and biotechnologytools(6).Morebroadly,the H2S Cu+ H detailed findings of this investigation provide foundational O S S knowledge of monocopper enzyme chemistry and the diversity of H Cys272 R biological reaction pathways for O2 activation.

Fig. 8. Proposed catalytic cycle of FGE inspired by this study. Materials and Methods Detailed procedures for protein purification, reagent synthesis, mass spec- trometry, Cu reconstitution, stopped-flow UV/vis absorption spectroscopy, resembles that proposed for the nonheme (II)-dependent enzyme activity assays, X-ray crystallography, size exclusion chromatogra- oxidase isopenicillin N synthase (IPNS). Calculations for IPNS phy–multiangle light scattering analysis, EPR spectroscopy, molecular dock- predict that an analogous thioaldehyde is formed at cysteine via ing and DFT computations, and XAS/EXAFS are provided in SI Appendix, SI – two 1e transfer steps from an inner-sphere ligated Fe-thiolate Materials and Methods. complex (31–33). From (4a)or(4b), dissociation and rapid hy- drolysis (34) of thioaldehyde (5) gives the final fGly product. At ACKNOWLEDGMENTS. We thank George Q. Fei for providing scFGE C272A/ this stage, FGE would undergo a two-electron reduction to C277A, and Dr. Stacy Malaker for assistance with mass spectrometry. This study return to its Cu(I) resting state (1). was supported by National Institutes of Health (NIH) Grant R01DK031450 (to E.I.S.), Ruth L. Kirschstein National Research Service Award F32GM116240 (to In the mechanism presented in Fig. 8, the requirement of ex- K.K.M.), and NIH Grant CA227942 (to C.R.B.). H.L. was supported by an Abbott ternal reducing agents would occur following product formation, Laboratories Stanford Graduate Fellowship. J.A.T. is supported by NIH Grant but this process remains speculative. Stoichiometric DTT oxidation – R35CA22043, a Robert A. Welch Chemistry Chair, and the Cancer Prevention during multiple turnovers of FGE (9) requires that 4e ultimately and Research Institute of Texas. See SI Appendix, SI Acknowledgements.

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