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Jun 29, 2017 - ABSTRACT: Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an import...
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Pulsed EPR insights into the ligand environment of copper in Drosophila lysyl oxidase Guodong Rao, Sandhya Bansal, Wen Xuan Law, Bing O'Dowd, Sergei A. Dikanov, and Eric Oldfield Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00308 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on June 29, 2017

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Pulsed EPR insights into the ligand environment of copper in Drosophila lysyl oxidase Guodong Rao,† Sandhya Bansal,† Wen Xuan Law,† Bing O'Dowd†, Sergei A. Dikanov,‡* and Eric Oldfield†*



Department of Chemistry, University of Illinois, Urbana, Illinois, 61801, United States



Department of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois 61801, United States

Corresponding Author *E.O.: [email protected], S.A.D: [email protected];

Funding This work was supported by the United States Public Health Service (National Institutes of Health Grant GM065307), by a Harriet A. Harlin Professorship (E.O.), and by the University of Illinois Foundation/Oldfield Research Fund. Pulsed EPR studies were supported in part by Grant DE-FG02-08ER15960 from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, US DOE (S.A.D.), and by NCRR/NIH Grants S10-RR15878 and S10-RR025438, for pulsed EPR instrumentation.

Notes The authors declare no competing financial interest.

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Abbreviations LOX, lysyl oxidase; EPR, electron paramagnetic resonance; ESEEM, electron spin-echo envelope modulation; HYSCORE, hyperfine sublevel-correlation; BAPN, β-aminopropionitrile; LTQ, lysine-tyrosyl-quinone;

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Abstract Lysyl oxidase is a copper amine oxidase that crosslinks collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development and formation of the "metastatic niche". Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, from Pichia pastoris). Here, we expressed active LOX from Drosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV-Vis, activity and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, ESEEM, and HYSCORE spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu-His interactions. At pH=9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH=7.5, there was evidence for a ~1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (β-aminopropionitrile; 1[13C15N]cyano-2-aminoethane) and the copper center—finding none. Overall, the results are of interest since they provide new spectroscopic information about the nature of the catalytic site in lysyl oxidase, an important anti-cancer drug target.

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Introduction Human lysyl oxidase (LOX, EC 1.4.3.13) is a copper protein that oxidizes lysine residues, presumably to the corresponding aldehyde, in primarily collagens and elastin. The aldehyde then crosslinks with the 6-amino groups in other lysines, resulting in non-enzymatic formation of the cross-links that are responsible for the mechanical strength and flexibility of these important structural proteins.1-5 LOX expression is regulated by hypoxia-inducible factors (HIFs) in cancer cells and is highly upregulated in many breast and lung tumors.6-10 Cox et al.11 have shown that LOX released by tumor cells under hypoxia has a major effect on osteoclastogenesis and bone remodeling, activating pre-osteoclasts that differentiate into active osteoclasts, and inhibiting osteoblasts. The result is the breakdown of bone homeostasis with significant bone destruction, leading to formation of the so-called "pre-metastatic niche". Compounds such as βaminopropionitrile (BAPN) and tetrathiomolybdate (MoS42-) are LOX inhibitors and have been found to inhibit metastases in mice models,12 as does LOX inhibition with a LOX antibody.13 Moreover, BAPN pro-drugs, in response to hypoxia, have proven to be effective in tumor growth inhibition.14 It has also recently been shown that LOX drives tumor progression by trapping epidermal growth factor receptors at the cell surface, an effect that is blocked by a small molecule LOX inhibitor.15 In addition, there is currently considerable interest in the development of other LOX and LOX-like inhibitors for both cancer and fibrosis therapeutics.3, 16, 17 Given its important roles in collagen and elastin crosslinking, and hence, angiogenesis, as well as its role in EGFR signaling, LOX is one of the most interesting amine oxidases. However, it is also the least characterized and perhaps the most difficult one to study experimentally, due to its low solubility in the absence of urea, and its low enzymatic activity. In early work, LOX was isolated from bovine aorta,18 human aorta19 and other biological samples.20, 21 Resonance Raman

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studies indicated the presence of an unique quinonoid cofactor, lysine-tyrosyl-quinone (LTQ),22 in native LOX, and continuous wave EPR spectra confirmed the presence of a copper (II) center.23 Later, human LOX and its analogues were heterologously expressed in E. coli and refolded from inclusion bodies,24 and the roles of several histidine residues were investigated by using site-directed mutagenesis.25 However, the concentration of soluble LOX obtained from such refolding protocols were too low (~10 µg/mL) to enable spectroscopic studies, and the activity of the refolded LOX was also low. In other work, Drosophila melanogaster lysyl oxidase-like protein (DmLOXL1), which shares a highly homologous catalytic domain with human LOX, was recombinantly expressed in native host Drosophila S2 cells, and more concentrated protein samples were obtained which enabled kinetic assays, as well as a spectroscopic investigation.26, 27 Based on the results outlined above, it is thus generally concluded that LOX catalyzes the oxidation of peptidyl amines with the LTQ cofactor plus a copper center, and proposed catalytic cycles for the formation of the LTQ cofactor, as well as for lysine side-chain oxidation, are shown in Figure 1.25 However, further biochemical characterization and mechanistic studies have been limited by the lack of any structural information. For example, the coordination environment of the copper site remains elusive, and has largely been proposed by analogy to other copper amine oxidases.28, 29. Additionally, it is not known how the BAPN inhibitor binds to the active site. Do the CN or amino groups coordinate to Cu? We thus sought to learn more about Cu-ligand (protein and inhibitor) interactions by using "modern" EPR spectroscopic methods. The application of EPR spectroscopy and related techniques for the study of copper proteins is an area of considerable interest.30, 31 Ligand coordination to Cu in proteins usually involves

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Figure 1. Proposed mechanism of formation of the LTQ cofactor and the catalytic cycle of LOX.

coordination of at least one histidine residue via one of the nitrogen atoms in the imidazole ring. This directly coordinated nitrogen (which can be Nτ or Nπ) has a hyperfine coupling constant of about 1.4 mT for 14N (~40 MHz), which can be detected in many cases in EPR and/or electronnuclear double resonance (ENDOR) experiments.32 Much smaller hyperfine coupling constants (about one-twentieth, i.e. ~2 MHz) are found for the remote imidazole nitrogen. These cannot be observed in CW-EPR spectra, e.g. of frozen solutions, and are also difficult to detect by using ENDOR spectroscopy. However, they are readily observed in X-band (~9.5 GHz) electron spin echo envelope modulation (ESEEM) spectra, as shown in the pioneering publications of Mims and Peisach in the 1970s.33-36 Since that time, ESEEM spectroscopy and later two-dimensional ESEEM, called HYSCORE, have been actively used for the study of copper coordination in the active sites of proteins.30, 37, 38 Although CW-EPR indicated the presence of a type II copper center in LOX,26 the number of His ligands and the identity of other possible residues were unclear, as was the nature of the interaction between the copper center and the LTQ cofactor, and the nature of the BAPN inhibitor-Cu interaction. Here, we thus sought to investigate some of these topics by using ESEEM and HYSCORE spectroscopies.

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Materials/Experimental Details General. All chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) unless otherwise indicated. Primers were synthesized by Integrated DNA Technologies (Coraville, IA). Restriction enzymes were from New England Biolabs (Beverly, MA). DNA purification kits were from Qiagen (Valencia, CA). MALDI-TOF MS, NMR and EPR experiments were performed in the corresponding facilities in the School of Chemical Sciences at the University of Illinois at Urbana-Champaign. Expression and purification of DmLOXL1. The plasmid containing DmLOXL1 was a kind gift from Dr. Katalin Csiszar (University of Hawaii at Mānoa). The transfection and protein expression protocols were basically as described previously,27 but modified to use a Ni-NTA affinity chromatography column. Briefly, the DmLOXL1 gene was cloned into a pMT/BiP/V5His vector (Fisher, MA) between the BglII and ApaI sites. The resulting plasmid was coprecipitated using calcium phosphate with a plasmid containing a hygromycin resistance marker. Drosophila S2 cells in complete medium were exposed to the precipitate for 24 h, then transferred to fresh medium with 500 µg/mL hygromycin. The cells were then cultured until stable transfected cells began to grow. For protein expression, recombinant cells were grown to 5×106 cells/mL in shake-flasks at 24 °C and induced with 0.5 mM copper sulfate. Cells were cultured for 2 more days before being removed by centrifugation. The medium was then diluted with an equal volume of water and 120 g/L urea containing 5 mM imidazole. Ni-NTA resin was added to the medium, and the suspension stirred for 60 min at 4 °C. The resin was collected by centrifugation, washed with TBS-2M urea (TBS is Tris-buffered saline: 50 mM Tris/HCl, 150 mM NaCl, pH=7.5) containing 10 mM imidazole, then eluted with TBS-2M urea containing 500 mM imidazole. Purified protein was dialyzed against TBS-2M urea containing 10 mM 2,2'-

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bipyridine for 1 day to remove metal ions, then against TBS-2M urea containing 0.5 mM CuSO4 for 3 days for copper incorporation, then against TBS-2 M urea containing 10 mM EDTA to remove adventitious Cu2+, and finally against TBS-2M urea buffer to remove EDTA. Activity and inhibition assays of lysyl oxidase. LOX activity was quantified by using an AmplexRed (10-acetyl-3,7-dihydroxyphenoxazine) fluorescence assay kit (Molecular Probes, OR) to monitor H2O2 formed during oxidation, according to the AmplexRed kit manual. Various amounts of recombinant DmLOXL1 in 100 µL assay buffer (TBS, 40 mM urea) were incubated with 10 mM lysine and the reaction monitored by using a fluorimeter. A series of H2O2 standard solutions (100 nM to 1 µM) were used to construct a calibration curve, and the specific activities of DmLOXL1 were calculated by using this curve. For the inhibition assays, 5 µg of DmLOXL1 were incubated for 10 min with different concentrations of BAPN (6-200 µM) prior to the activity assay. Does-response curves were fit by using OriginPro 2016 (OriginLab, Northampton, MA) software. Synthesis of the TFA salt of (1-13C, 15N)-BAPN. A mixture of 2-(boc-amino) ethyl bromide (500 mg, 2.23 mmol) and K13C15N (210 mg, 3.13 mmol) in DMF (10 mL) was stirred overnight at room temperature. The reaction was then diluted with DCM and washed with water, dried over Na2SO4 and concentrated. The crude nitrile was purified using flash chromatography (hexane: ethyl acetate = 20:1 to 6:1) as a light yellow oil. To a cold solution (0 °C) of the nitrile in 3 mL DCM was added TFA (1.5 mL), dropwise. The mixture was stirred at 0 °C for 2h and then concentrated into an orange oil. DCM was added to induce precipitation. The solid was then filtered, washed with DCM, and recrystallized from methanol/ether to afford the TFA salt of (113

C,

15

N)-BAPN as a white solid (130 mg, 32%). 1H NMR (500 MHz, CD3OD): δ 2.88 (-

CH2CH213C15N, ddt, 3J15N = 1.5 Hz, 2J13C = 10.5 Hz, 3JH = 6.5 Hz, 2H), 3.26 (-CH2CH213C15N, dt,

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3

J13C = 6.0 Hz, 3JH = 6.5 Hz, 2H). 13C NMR (125.7 MHz, CD3OD): δ 16.6 (-CH213C15N, dd, 1J13C

= 57.9 Hz, 2J15N = 2.8 Hz), 36.7 (-CH2CH213C15N), 117.5 (-13C15N, d, 1J15N = 17.5 Hz). 15N NMR (CD3OD, 50.7 MHz, NH3 = 0 ppm): δ 249.5 (d, 1J13C = 17.2 Hz). HRMS (ESI+) calcd for C2H7N13C15N [M + H] +, 73.0608; found, 73.0607. EPR Sample preparation. Cu(imidazole)n (n=1, 2, 4) samples were made as following: for n=1, 10 µL 100 mM CuSO4, 10 µL 100 mM diethylenetriamine, and 10 µL 100 mM imidazole were mixed together and diluted to 1 mL with 50% glycerol; for n=2, 10 µL 100 mM CuSO4, 10 µL 100 mM ethylenediamine, and 20 µL 100 mM imidazole were mixed together and diluted to 1 mL with 50% glycerol; for n=4, 10 µL 100 mM CuSO4 and 60 µL 100 mM imidazole were mixed together and diluted to 1 mL with 50% glycerol. Sample identities were confirmed by comparison to reported CW-EPR spectra.39 For protein samples, DmLOXL1 was concentrated to ~0.2 mM. Glycerol was added to 25% as a glassing agent. β-aminopropionitrile was added to 5 mM, as needed. All samples were transferred to EPR tubes, frozen, and stored in liquid nitrogen prior to use. CW- and pulsed EPR. CW-EPR experiments were performed at X-band (9 GHz) on a Varian E-122 spectrometer using an Air Products (Allentown, PA) helium cryostat. CW-EPR spectra were recorded at non-saturating conditions. Pulsed-EPR experiments were performed on a Bruker ELEXSYS E-580-10 FT-EPR spectrometer with an Oxford CF935 cryostat. All pulsed experiments were performed at 30 K. Electron spin-echo field swept used a pulse sequence π/2τ-π-τ-echo and τ = 136 ns. 2-pulse ESEEM used a pulse sequence π/2-τ-π-τ-echo with a τ0 of 100 ns. 3-pulse ESEEM used a pulse sequence π/2-τ-π/2-T-π/2-τ-echo with variable T. HYSCORE experiments used the sequence π/2-τ-π/2-t1-π-t2-π/2-τ-echo with τ=136 ns and 256 points for both t1 and t2 with 16 ns steps. The π/2 pulse length was 16 ns in all pulse experiments. For pulse

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experiment data processing, time domain data were baseline-corrected with a third order polynomial, Hamming-window function multiplied, zero-filled, Fourier-transformed and visualized by using Matlab (R2014a). Simulations of the EPR spectra (as shown in the Supplementary Material) were performed by using the Easyspin toolbox (5.1.3).40

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Results and Discussion LOX family proteins and conserved residues. Human LOX and LOX-like proteins all contain a conserved C-terminal catalytic domain that harbors the LTQ cofactor and the copper site,4 Figure 2A. LOX is also conserved among many other animals and a similarity search shows that LTQ-containing LOX is also likely to be present in some bacteria (myxobacteria and actinomyces) and fungi, Supporting Information Figure S1—although the function of these putative LOXs is unknown. Please note that human LOX is not to be confused with the trihydroxyphenylalanine-quinone (TPQ) containing "LOX" found in Pichia pastoris41 (PDB ID code 1N9E42) which is approximately twice as large, has no sequence identity, and is in fact structurally very similar to a different human protein, diamine oxidase (PDB ID code 3MPH43), with a 2.1 Å Cα root mean square deviation between the 2 structures.

Figure 2. The LOX family proteins. (A) The domain architecture of LOX from different organisms. Adapted from Ref. 4. (B) Conserved residues in human LOX via SCORECONS.

Since there is basically no structural information on human LOX, and we were not successful in generating high quality structural models using Phyre244 or I-TASSER,45 we next determined the conserved residues in LOX and LOXL proteins by using the SCORECONS program.46

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Results are shown in Figure 2B. The conserved Tyr355 and Lys320 that form the LTQ cofactor are present, as expected, as are two conserved His, His294 and His296, which could coordinate to the copper center. These conserved residues are also present in LOX homologues from e.g. Spizellomyces punctatus and Enhygromyxa salina, as seen in the sequence alignment in Figure S1. An interesting question is, therefore, how many His ligands are there in the active site of lysyl oxidase? Previous studies suggested that H292, H294 and H296 are the three His ligands for the copper center.25 However, only H294 and H296 are highly conserved His residues, according to the SCORECONS results. In addition, while the “HXH” copper binding motif is found in the X-ray structures of numerous copper containing proteins,41,

47-49

the “HXHXH”

motif is not found. Instead, when three His are coordinated to copper, the third His is usually ~150 residues distant.41, 48 In the case of LOX, such a conserved remote His residue is not observed. This raises the possibility that there are actually 2 His ligands, H294 and H296, at the copper center, as opposed to the situation found in the TPQ-containing LOX from Pichia pastoris.41 There are also several other residues that are highly conserved: two Cys (C324, C340); two Asp (D353, D365); a Gln (Q362) and a Tyr (Y374). While the roles of these residues remain to be determined, they could be involved in disulfide bond formation, copper binding, or in a proton transfer network. We thus next expressed DmLOXL1 in order to investigate its activity and possible Cu-ligand (protein, inhibitor) interactions. Expression and biochemical characterization of lysyl oxidase. Attempts to express human LOX and LOX-like proteins in E. coli using previously reported protocols25, 50 generally yielded inclusion bodies that cannot be refolded into active proteins. We thus expressed the highly homologous and soluble Drosophila melanogaster LOX-like protein, DmLOXL1, in Drosophila S2 cells and purified it to homogeneity. The identity of the protein was confirmed by MS

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(calculated: 45411, actual: 45460). Incorporation of copper was performed by using a series of dialysis steps yielding the final mature LOX as a light pinkish-brown protein that showed a broad absorption at 500-520 nm, Figure 3A. After adding phenylhydrazine, the sample turned yellow and exhibited a much stronger absorption peak at ~450 nm, Figure 3A, indicating the presence of the quinone cofactor, consistent with previous reports.26 We then measured the activity of DmLOXL1 by using the AmplexRed fluorescence assay kit, which detects H2O2 formation during amine oxidation. We used lysine as substrate since lysine is the residue that is oxidized by LOX. The activities of LOX were proportional to enzyme concentration, Figure 3B. The initial specific activity of DmLOXL1 was 23.0 µmol/mg/min at 37 °C in the presence of 10 mM lysine, Figure 3B. This value is much higher than the previously reported activity value with lysine, 50 nmol/mg/min.26 The activity of DmLOXL1 was inhibited by BAPN, the known LOX inhibitor, in a dose-dependent manner with an IC50 of 22 µM (Figure 3C). These results indicate that we have a correctly folded, highly active and inhibitable DmLOXL1 that is suitable for further studies.

Figure 3. Biochemical characterization of DmLOXL1. (A) UV-Vis of DmLOXL1 and its phenylhydrazine adduct. (B) Kinetics assay of DmLOXL1 with different amount of enzyme. (C) Doseresponse curve of the inhibition of DmLOXL1 by BAPN.

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EPR of DmLOXL1. We next investigated the copper site of DmLOXL1 by using EPR spectroscopy. One EPR spectrum of DmLOXL1 has been reported previously, but since neither pH values nor simulations were reported and the samples had low activity, we first obtained the CW-EPR spectrum of active DmLOXL1 at pH=7.5, shown in the blue trace in Figure 4. Clearly, there are two species present with similar intensity, shown by the splitting of the peak at B~275 mT. In addition, a poorly resolved splitting, ~40 MHz, can be seen in the g⊥ region of the spectrum (Figure S2), mostly likely originating from the directly coordinating nitrogen on the His ligands.51 It has been noted in an earlier EPR study of bovine aortic LOX23 that only one species was dominant at higher pH and consistent with this, the CW-EPR spectrum of DmLOXL1 at pH=9.3, green trace in Figure 4, is dominated by one species (species 1), but there are clearly two components in the pH=7.5 spectrum. We next simulated both spectra using EasySpin40 using the following principal values of the g tensor and A// copper (63Cu) hyperfine coupling: species 1, g = [2.305, 2.066, 2.065], and A// (63Cu) = 465 MHz (153×10-4 cm-1; 14.4 mT); species 2, g = [2.265, 2.075, 2.065], and A// (63Cu) = 415 MHz (153×10-4 cm-1; 14.4 mT). The ratio between species 1 and species 2 was 1.1:1 (52%:48%) in the pH=7.5 sample, and 6:1 (86%:14%) in the pH=9.3 sample. The speciation of DmLOXL1 at the different pH values is

Figure 4. CW-EPR of DmLOXL1 at pH=7.5 (blue trace), pH=9.3 (green trace) and their simulations (red). The signal at g=2.00 is from the contribution of a small amount of His-tag bound copper species. Typical parameters for recording CW-EPR were as follow: temperature, 15K; microwave frequency, 9.38 GHz; microwave power: 0.02 mW; modulation amplitude, 0.5 mT. See SI for spectral simulations.

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also revealed in the corresponding field-swept electron spin-echo detected absorption spectra (Figure S3). Table 1. Spectroscopic properties of Type II copper centers with His ligands. Copper centers dopamine β-hydroxylase laccase ascorbate oxidase galactose oxidase amine oxidase DmLOXL1 species 1 DmLOXL1 species 2 CuIm1 CuIm2 CuIm4

g// 2.27 2.24 2.24 2.277 2.30 2.295 2.264 2.212 2.228 2.242

A//, (Cu)a 450 618 606 558 489 465 415 575 570 583

NQIa 0.7, 0.7, 1.4 0.59, 0.8, 1.48 0.61, 0.89, 1.54 0.61, 0.89, 1.54 0.45, 1.1, 1.55 0.44, 1.10, 1.54 0.74, 0.89, 1.63 0.54, 0.88, 1.47 0.53, 0.96, 1.50 0.42, 1.05, 1.47

e2Qq/ha 1.44 1.52 1.56 1.56 1.73 1.76 1.68 1.56 1.64 1.68

η 0.98 0.89 0.83 0.83 0.48 0.50 0.89 0.69 0.65 0.50

aisoa 1.75 N/A N/A N/A 2.3 1.90 1.60 1.74 1.74 1.74

ligands 3His+2Hisb 3His 2His 2His 2Tyr 3His 1His 3His 1Imid 2Imid 4Imid

ref [63] [52] [64] [65] [66] This study This study This study This study This study

a. MHz; b. Two Cu sites are present, one with three His ligands and the other two His.

Both species 1 and species 2 possess the typical axial anisotropy of tetragonally (elongated) copper complexes with a ݀୶మ ି௬మ ground state, and g//>g⊥>ge.52 The g// and A// (Cu) values for these two species and some other type II copper sites are summarized in Table 1. Species 1 has a copper A// value of 460 MHz, typical for type II copper centers,52 while species 2 has an A// of 405 MHz, ~10-20% lower than usual for a type II copper, indicating a larger spin delocalization and hence, higher covalency at this copper site. Since the protein was purified via His-tag affinity chromatography, we were concerned that His-tag-bound copper might be one of these species. However, this possibility was ruled out by comparing the EPR spectra of DmLOXL1 to a sample in which His-tag bound copper was the dominant species (see Figure S4 for details). The g// and A// for this His-tag bound copper did not match either of the two LOX species. It is therefore likely that both species are copper signals bound to the active site of DmLOXL1. The question then arises as to what are the differences between the coordination environments of these two species? ESEEM of DmLOXL1 in comparison with CuImn complexes. We next used ESEEM spectroscopy to try and characterize the interactions between the Cu(II) center and the remote

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Figure 5. (A) CW-EPR spectra of CuImn (n=1, 2, and 4) and DmLOXL1 at different pH values. (B) Time domain 2-pulse ESEEM patterns of CuImn and DmLOXL1 recorded at g~2.06. (C) Time domain 2-pulse ESEEM patterns of CuImn and DmLOXL1 recorded at g~2.43. Parameters for recording CWEPR were the same as those in Figure 4.

nitrogen of the coordinated imidazole ring(s) in order to try and deduce the number of His ligands in the two species. We obtained ESEEM results for DmLOXL1 as well as for model Cu (II)-imidazole complexes (CuImn) containing n=1, 2 and 4 imidazole ligands. This approach has previously been employed in studies of many copper proteins.39, 53, 54 CuIm3 complexes have never been used though, probably because model systems are not available since simple CuIm3 complexes are likely to disproportionate into more stable Im2 plus Im4 complexes. The identities of these small molecule complexes (in frozen samples) were confirmed by CW-EPR, and are to be compared with the CW-EPR spectra of the two DmLOXL1 samples, Figure 5A. 2-pulse ESEEM patterns of the CuImn models as well as the DmLOXL1 samples at different pH values, recorded at g⊥ (the point of maximum signal intensity, g~2.06) and g// (lowest magnetic field, g ~ 2.43) are shown in Figures 5B and 5C, respectively. What the ESEEM results show is that the modulation depth of the low frequency ESEEM from remote nitrogens increases monotonically with an increasing number of imidazole ligands,34 leading to an increasingly smaller distance

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from the points with minimal echo amplitude to the baseline. At the same time, the amplitude ratio between the low frequency nitrogen modulation to the high frequency (~14 MHz) proton modulation, increases. On inspection of the DmLOXL1 results it appears that the pH 7.5 sample exhibits the most similar modulation-depth and time-domain patterns to those found in CuIm2— but in the CW spectrum we clearly have a 2-component system—while the pH 9.3 sample is most similar to CuIm1, corresponding to one His ligand bound to Cu in the major species at this pH (species 1). It thus seemed likely that the apparent “CuIm2" similarity might actually be due to the presence of a ~1:1 mixture of CuIm1+CuIm3 species (see the notes in SI), the tri-His coordination being found in several Cu proteins as well as having been proposed to occur in LOX.25, 26

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Figure 6. Three-pulse ESEEM spectra of CuImn (n=1, 2, and 4) and DmLOXL1 samples at g ~ 2.06 with τ=140 ns (A) and 100 ns (B). The 14N and 1H ESEEM intensities are shown in (B) as the integration of the corresponding regions, as described in the Text. (C) Linear fit of the relative intensities of 14N-ESEEM versus the number of imidazole ligands together with the predicted average number of Cu-His ligands in different DmLOXL1 samples.

In order to obtain additional information on possible Cu-His interactions, we investigated the 3-pulse ESEEM of the CuImn and DmLOXL1 samples, this time in the frequency domain. Background information on the technique is given in the Supporting Information. Spectra collected at the field with maximum EPR signal intensity (g ~ 2.06) are shown in Figure 6A. In this region, the different imidazole orientations contribute to the echo signal and permit use of spectral analyses originally formulated for orientation-disordered samples.30 In agreement with previous observations,39,

53, 54

all of the CuImn complexes exhibit spectra typical of "exact

cancellation". Specifically, they contain a sharp ν+ peak at ~1.5 MHz, and a broad νdq line of varying intensity, at ~4 MHz. Two other pure quadrupole peaks, ν0 and ν-, have lower intensity and are poorly separated from each other, indicating that the asymmetry parameter η in these complexes is close to 1 (Eq. S1). The intensity of the broad νdq peak increases relative to that of the sharp ν+ peak in the model complexes as the number of imidazole ligands increases.55 In

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addition, the νc ~ 2.6 MHz peak that is seen in the spectrum of CuIm2 can be interpreted as a part of the unresolved combination features—including ν+ and ν-,0, nuclear quadrupole frequencies that are within the same electron manifold—due to the presence of multiple imidazole ligands.5658

The intensity of the νc peak is significantly increased in CuIm4, and is also accompanied by the

appearance of a broad peak with low intensity at ~8.5 MHz, close to the 2νdq combination frequency,55, 59 due to the presence of more imidazole ligands in the complex.52, 56 The 3 pulse-ESEEM spectra of DmLOXL1 at pH=7.5 also exhibit a lineshape indicative of cancellation, with a sharp ν+ peak at ~1.5 MHz, and a broader νdq at ~4 MHz. The combination peak νc at ~2.6 MHz is also clearly seen, suggesting the presence of multiple His ligands. However, the quadrupole peaks in the DmLOXL1 samples in general have broad linewidths, due to much faster ESEEM decay in the time domain (see Figure S5 for the time domain patterns). Because of this, and most likely an η close to 1, the two quadrupole peaks, ν0 and ν-, are not distinguishable in the protein sample. The spectrum of the pH=9.3 sample has a more obvious separation between ν0 and ν-, suggestive of a rather different asymmetry parameter. In addition, no obvious combination peak could be observed (and the contribution from ~ 14% of species 2 is small), consistent with our previous conclusion that only a single His ligand is bound to Cu in the major species at this higher pH value. We also obtained the three-pulse ESEEM spectra in the gz region (g ~ 2.43), Figures S6. Orientation-selected ESEEM spectra of the model complexes, which are close to the “single-crystal-like” condition, show better resolution of the ν‒ and ν0 lines in the nuclear quadrupole triplet, and there are additional splittings of the νc peak, at 2.1 and 2.5 MHz, corresponding to (ν+ + ν0) and (ν+ + ν-) combinations. However, the signal intensities for the protein samples were too weak to obtain useful information. We simulated the frequency domain of the 3p-ESEEM spectra for the CuImn complexes and the protein samples

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(Figure S7), and extracted the

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N nuclear quadrupole and hyperfine parameters for these

samples. These parameters, as well as the values for some other type II copper centers, are shown for comparison in Table 1. The hyperfine and quadrupole parameters in DmLOXL1 are all comparable to other type II copper centers with His ligands. To try and better determine the number of His ligands in species 1 and species 2 in the DmLOXL1 samples, we next utilized the method described in a recent report39 in which the number of imidazole ligands near the copper center was determined by comparing the relative intensities of the

14

N-ESEEM peaks. Specifically, the

14

N-ESEEM intensity is calculated by

integrating the peaks in the 0-10 MHz region of the frequency domain of the three-pulse ESEEM, and is then divided by the 1H-ESEEM intensity between 13-16 MHz in the same spectrum. The resulting

14

N to 1H integrated intensity ratios were shown to be proportional to the number of

imidazoles coordinating Cu. Following this approach and using τ=100 ns (where the 1H ESEEM signal is not suppressed, Figure 6B), we obtained the integrated areas for 14N, 1H and their ratios for the 3 different model complexes, shown plotted as a function of the number of imidazole ligands in Figure 6C (and constrained to pass through the origin). As expected, there is an excellent correlation between the ESEEM intensity ratios and the number of imidazole ligands in the model compounds, and when using the protein data we find (red circles, Figure 6C) that the pH 7.5 sample (a mixture) appears to have very close to 2 imidazoles (i.e. histidines) on average, while the pH 9.3 sample has very close to 1. What these results strongly suggest then is that—as alluded to above—a single His binds to Cu at pH=9.3, while at pH=7.5 there is a ~1:1 mixture of 1 and 3 His-ligated proteins. Since LOX is thought to contain just one copper in its active site, these two species are likely to arise from a pH-dependent ligand displacement, resulting in

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dominance of the single His-ligated species (which is ~0.5 kJ more stable than the 3 His species) at the basic pH value. The assignments of the frequencies in the three-pulse ESEEM spectra of DmLOX1 are supported by the results of a two dimensional HYSCORE experiment (Figures 7A, B and S8A, B). The spectra of both samples exhibit cross-peaks 1 and 2 correlating ν+ and ν−~ν0 with νdq+, respectively, confirming that these spectral features originate from different electron spin manifolds. These correlations also permit a more precise determination of the value of νdq+ ≈ 4.3−4.7 MHz from the maximum of the most intense (νdq+, ν+) cross-correlation. Cross-peaks 1 and 2 are also oriented parallel to the coordinate axis and there is negligible orientation dependence of the ν+, ν−, ν0 frequencies (equal to the pure nuclear quadrupole resonance frequencies) as result of a cancellation condition in the corresponding spin manifold.60 Moreover, cross-peaks 3 that correlate the combination frequency νc with νdq+ can only be seen in the pH=7.5 sample. This observation confirms that there are multiple His ligands in this sample, while in the pH=9.3 sample, only one His ligand is present. In addition, spectra of the pH=7.5 sample show that cross-peaks 4, of low intensity and oriented approximately normal to the

Figure 7. HYSCORE spectra of DmLOXL1 samples. (A) X-band HYSCORE spectrum of DmLOXL1 at pH=7.5. (B) X-band HYSCORE spectrum of DmLOXL1 at pH=9.3. (C) X-band HYSCORE spectrum of DmLOXL1+[1-13C,15N]-BAPN. Microwave frequency 9.63 GHz, magnetic field 333 mT. τ = 136 ns for all spectra. 21

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diagonal, are likely to originate from νdq+, νdq- from other type of nitrogen with higher quadrupole coupling constant.60 Plus, simulations of the 2p-ESEEM spectra (Figure S9) are consistent with this 2-component model. EPR of BAPN bound DmLOXL1. There is considerable interest in developing inhibitors for LOX since they could be new cancer therapeutic leads.61, 62 BAPN is a known LOX inhibitor, but its use is limited by toxicity. However, it is still of interest to understand the molecular mechanism of action of BAPN since this could facilitate mechanism-based inhibitor development. BAPN has been proposed to covalently link to the active site of LOX, presumably to the quinone. The nitrile moiety on BAPN might also bind to copper, if the copper center is close to the quinone cofactor. In order to investigate possible interactions between the nitrile group of BAPN and the copper center, we obtained 2-pulse and 3-pulse ESEEM spectra of BAPN bound DmLOXL1 and compared them to spectra of the protein-only sample, Figures S10A, B. The 2-pulse and 3-pulse ESEEM spectra are almost the same for ligand-free and inhibitor- bound protein samples, except for a small peak at ~4.9 MHz seen in the frequency domain 3-pulse spectrum, Figure S10B. In order to further examine any weak interactions, we synthesized BAPN with a 13C15N nitrile group, and measured HYSCORE spectra of DmLOXL1 with and without the labelled BAPN (Figures 7C, S8C,D). After incubating with BAPN, there was the expected color change from pink to light yellow, as seen with the phenylhydrazine adduct, implying formation of a Schiff-base between the quinone cofactor and BAPN. In the HYSCORE spectra, however, there were no obvious

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C or

15

N hyperfine couplings observed,

Figures 7C and S8C, D. Features 1, 2 and 3 in the HYSCORE spectra remained largely unaffected, but feature 4 was much weaker in the BAPN-bound spectrum, due perhaps to a conformational change in the surrounding residues on BAPN binding. These results indicate then,

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that BAPN, or at least its nitrile group, is relatively far away (>6 Å since the

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C hyperfine

dipolar interaction is