Protonation and Sulfido versus Oxo Ligation Changes at the

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Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X‑ray Absorption Spectroscopy Stefan Reschke,† Stefan Mebs,‡ Kajsa G. V. Sigfridsson-Clauss,‡,§ Ramona Kositzki,‡ Silke Leimkühler,*,† and Michael Haumann*,‡ †

Institut für Biochemie und Biologie, Molekulare Enzymologie, Universität Potsdam, 14476 Potsdam, Germany Institut für Experimentalphysik, Freie Universität Berlin, 14195 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5− 10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo(VI) ion with each one MPT, MoO, Mo−O−, and MoS ligand, and a weak Mo−O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo−OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.

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INTRODUCTION Enzymes containing a molybdenum cofactor (Moco) catalyze a broad range of essential substrate oxidation reactions in all kingdoms of life.1−7 Xanthine dehydrogenase (XDH) (also denoted xanthine oxidoreductase) belongs to the same family of molybdoenzymes as aldehyde oxidase (AOX).8−11 These enzymes show a similar homodimeric structure, with an Nterminal domain containing two [2Fe-2S] clusters serving as electron transfer relays between a flavin adenine dinucleotide molecule in the central domain and the Moco bound to the Cterminal domain, and evolutionary trace back to a common ancestor.3,11−13 XDHs and AOXs can form reactive oxygen species14,15 and therefore are interesting medical targets.16−20 Understanding structure−function relations at the Moco during catalysis in this enzyme family thus may have an impact on human health applications.21,22 X-ray crystallography has established that the Moco of XDHs consists of a molybdenum ion, which is coordinated by the two dithiolene sulfurs of a single molybdopterin ligand (MPT) (Figure 1).1,11,23−25 Crystal and spectroscopic studies further have suggested that in the catalytically competent Moco in XDH and AOX, the Mo coordination is complemented by two © XXXX American Chemical Society

oxygen ligands and a terminal sulfido group (MoS binding motif) in a distorted square-pyramidal geometry (Figure 1 and Table S1).9,11,24,26−33 In particular, the involvement of sulfido ligands in molybdoenzme catalysis has attracted much research interest because of their ability for protonation changes.9,34−37 For the XDH from the bacterium Rhodobacter capsulatus (XDHRc), it has been established that the Moco binding chaperone XdhC is required for the insertion of the sulfido ligand at the molybdenum ion.38−40 Several conserved amino acids in the vicinity of Moco interfere with catalytic activity in XDHs.41−44 The carboxylic side chain of Glu730 in XDHRc is as close as 2.7 Å to the metal in crystal structures and may function as a proton acceptor during catalysis, whereas the amino group of Gln179 may affect the cofactor properties by hydrogen bonding interactions with the metal ligands23,41,45,46 (Figure 1). In XDH and related AOX enzymes, it has been revealed that a substrate molecule coordinates at the Mo center via the oxygen atom at position 2 (Figure 1).45,47−50 The sulfido group Received: November 24, 2016

A

DOI: 10.1021/acs.inorgchem.6b02846 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

nitrilotriacetic acid chromatography, by Q-sepharose chromatography, and by additional folate affinity chromatography (the latter was used for only WT and Q197A). Protein concentrations were determined from the absorbance at 465 nm (ε = 31.6 mM−1 cm−1). The metal loading of purified enzymes was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) on a PerkinElmer (Fremont, CA) spectrometer using the multielement standard solution XVI (from Merck) as a reference.65 Protein samples were incubated overnight in a 1:1 mixture with 65% nitric acid (Suprapur, Merck, Darmstadt, Germany) at 100 °C and diluted [10:1 (v/v)] with ultrapure water (Millipore) prior to ICP-OES analysis. Activities of enzyme variants were normalized to molybdenum contents.43 Proteins were transferred to ultracentrifugation devices (VIVASPIN 20, 50 kDa cutoff; Sartorius, Göttingen, Germany), concentrated to ∼1 mM enzyme-bound Mo, and thereafter transferred to sample holders (30 μL) for XAS and stored in liquid nitrogen. Activity Studies. Steady-state enzyme kinetic analysis of xanthine oxidation was performed as previously described43,46 at 25 °C in 1 mL cuvettes in a reaction mixture containing 5 μM (0.675 mg mL−1) XDH proteins and 100 mM CAPS buffer with 100 mM KCl (pH 9−10), 50 mM Tris buffer with 100 mM NaCl (pH 7−9), or 100 mM MES buffer with 100 mM KCl (pH 5.5−6.5) using 50 mM NAD+ as an electron acceptor and increasing xanthine (Sigma-Aldrich) concentrations (from 25 to 250 μM). The reaction was followed for 1 min via the increase in absorption at 340 nm due to NADH formation with a spectrophotometer (Shimadzu UV-2401PC). From the absorption traces (triple determinations for each xanthine concentration and pH value), reaction velocities were derived and analyzed in the frame of the Michaelis−Menten theory for determination of apparent Vmax and KM values. pH-dependent activities were analyzed using eq 1 for pK determination (Amax, maximal activity):

Figure 1. Crystal structure of the molybdenum cofactor in XDH. The structure is that of the R. capsulatus enzyme (Protein Data Bank entry 1JRO, 2.7 Å resolution);28 discrimination of oxygen or sulfur ligands in apical or equatorial positions at the Mo ion has not been attempted. MPT represents molybdopterin; side chains of amino acid residues Gln179 and Glu730 are at the indicated distances (in angstroms) from molybdenum and oxygen atoms (dashed lines).

was proposed to act as a proton acceptor during the twoelectron reduction of the initial Mo(VI) to a Mo(IV) species and oxidation and dehydration of the (xanthine) substrate to form the (uric acid) product.10,23,27,28,45,48 The now commonly proposed mechanism for xanthine oxidation involves baseassisted nucleophilic attack on the substrate by a Mo(VI)bound hydroxo (OH) group with concomitant hydride transfer to form a Mo−SH group and the Mo(IV)−O−R product (see, e.g., refs 1, 10, 11, 17, 23, 47, and 48 for review). The reaction path is closely related to the protonation state of sulfur and oxygen species at the active site. Most crystallographic and spectroscopic studies of XDH and AOX enzymes have positioned the sulfido group in an equatorial position and an oxo group in an apical position at molybdenum (Figure 1 and Table S1)9,11,24,26−33,45,47−50,52−56 (see refs 49 and 51 for an alternative ligand arrangement). An equatorial selenido group was found in a crystal structure of nicotinate dehydrogenase.37 Earlier X-ray absorption spectroscopy (XAS) studies of XDH enzymes have suggested a MoS group and two oxo ligands at Mo at alkaline pH and a protonation change at acidic pH.31,57−63 The fact that the enzymes are largely inactive at acidic pH11,43 may qualify a role of an initially present hydroxo group at the oxidized metal in catalysis. Here, we used XAS at the Mo K-edge to determine the molybdenum coordination in wild-type (WT) XDHRc and in the E730A and Q179A variants in a wide pH range (5−10). Of the two oxo ligands at the metal found at alkaline pH, one was protonated to a Mo−OH group with a similar pK of ∼6.8 in WT and Q179A. This protonation and replacement of the sulfido with an oxo ligand in E730A inactivated the enzyme. Density functional theory (DFT) calculations on Moco model structures best reproduced the bond lengths from XAS for equatorial MoS and Mo−O− ligands at Mo(VI). These findings suggest that an initial oxo rather than a hydroxo group at the metal is important in substrate oxidation by XDHRc.

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A = A max [(1 + 10 pH − pK1)−1 − (1 + 10 pK2 − pH)−1]

(1)

X-ray Absorption Spectroscopy. XAS at the Mo K-edge was performed at the SOLEIL synchrotron (Paris, France) at the SAMBA beamline using a Si[220] double-crystal monochromator and a 36element energy-resolving Ge detector (Canberra) for X-ray fluorescence detection in a conventional XAS setup.36,66,67 Protein samples were held in a liquid helium cryostat at 20 K. Calibration of the energy axis was performed using the absorption edge of a Mo foil as a standard (reference energy of 20003.9 eV).67 Up to 10 XAS scans (∼20 min duration) per sample (each on a fresh spot) were averaged for signal-to-noise ratio improvement. Normalized XANES and EXAFS spectra were derived from energy scan data as described previously.68 EXAFS simulations were performed with the in-house software SimX and phase functions calculated by FEFF9.69 Fourier transforms of EXAFS spectra were calculated for a k range of 1.6−14.1 Å−1 (cos windows extending over 10% of both k range ends); S02 was 1.0. Coordination number (N) and interatomic distance (R) variations as a function of pH were fitted by titration curves (analogous to eq 1, but including an additional offset value and only one pK term). Bond valence sum (BVS) calculations (eq 2) included N and R values from EXAFS analysis (the sum is over all Mo−ligand bonds). The B value was 0.37 Å; R0 values for Mo(VI) were as follows: 1.87 Å for Mo−O and 2.33 Å for Mo−S.70−73

BVS =

⎛ R0 − Ri ⎞ ⎟ B ⎠

∑ Ni exp⎜⎝

(2)

Density Functional Theory (DFT) Calculations. DFT calculations were performed on truncated cofactor models (Figure S1) initially derived from an XDHRc crystal structure (Protein Data Bank entry 1JRO). Amino acids were truncated and saturated with protons, and structures including Mo(VI) were geometry-optimized by DFT, fixing the outer C atoms of the amino acid groups, in a COSMO solvation environment (ε = 38) using Gaussian0974 and the B3LYP/ TZVP functional/basis set combination for all atoms except Mo, for which an ECP8MDF/VTZ(cc-pVT2-PP/VTZPP) effective-core-potential functional/basis set was used.75,76 Protons were added in the

MATERIALS AND METHODS

Enzyme Purification and Sample Preparation. Wild-type XDH from R. capsulatus (WT) and its E730A and Q197A variants were purified after heterologous expression in Escherichia coli TP1000 as described previously.64 Protein purification was performed via nickelB


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Inorganic Chemistry structures at Mo ligands in equatorial or apical positions. For further details, see Figure S1.

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RESULTS Enzyme Kinetics. We characterized XDHRc protein representing the wild-type enzyme (WT) and variants in

Figure 2. Enzyme kinetics of XDHRc. (A) Xanthine oxidation activity (Vmaxapp/KMapp) plotted vs the sample pH for WT and Q179A (symbols) from kinetic data analysis (eq 3 and Figure S2). The solid red line is the fit curve (eq 1) with the indicated pK values; dashed blue and green lines are pH titrations for the individual pK values. (B) pH dependence of experimental apparent Vmax and KM values for the two variants (symbols) and calculated data (lines) for the two inhibitory branches in the scheme in panel C for Vmaxapp (green lines) and KMapp (blue lines) (solid lines, eq 4; dashed lines, eq 5), derived from calculated kinetic data (Figure S4) using intrinsic values for KM of 0.058 mM and Vmax of 115 s−1. (C) Enzyme kinetic scheme according to Michaelis−Menten theory (black, eq 3) and including noncompetitive (green, eq 4) or competitive (blue, eq 5) inhibition paths depending on protonation events with two pK values (E, enzyme; S, substrate; P, product; H, protonation; k1−3, rate constants; KI, equilibrium constant for inhibitor binding).

Figure 3. XAS analysis of wild-type XDHRc. (A) XANES spectra at three pH values. The pre-edge feature (asterisk) is magnified in inset a. Inset b shows the pre-edge amplitudes (20009.5 eV) for 20 WT samples (■) in the pH range of 5.0−10.0 and a fit curve with a pK of 6.8. Empty triangles show data for Q179A. (B) Fourier transform (FT) of the experimental EXAFS spectrum of WT (pH 7.0) and fit curve with parameters in Table 1, Fourier-filtered (ff, FT range of 1.0− 3.5 Å) experimental and simulated spectra, and metal ligand contributions to the fitted EXAFS (vertically displaced), top to bottom, respectively. The inset shows the respective experimental and calculated EXAFS oscillations. (C) FTs of WT at three pH values for EXAFS data in the inset (black lines, experimental data; colored lines, fits with parameters in Figure 5 and Table 1).

which alanine replaced Glu730 (E730A) or Gln179 (Q179A). Xanthine oxidation rates were determined in a spectrophotometrical assay, and reaction velocities (V) were derived from the kinetic transients for increasing substrate concentrations ([S]) in a pH range of 5.5−10.0 (Figure S2). Apparent maximal velocities (Vmaxapp) and substrate binding constants (KMapp) were obtained from data fitting using the standard Michaelis− Menten relation (eq 3).77 The analysis results are shown in Figure 2. WT showed maximal activity (Vmaxapp/KMapp) at pH ∼8.0, and the bell-shaped activity decrease at more acidic or

alkaline pH was described (eq 1) with pK values of 6.8 ± 0.1 and 8.8 ± 0.1. These pK’s were similar to previously determined values for WT XDHRc.43 The Q179A variant showed a similar activity profile and pK values that, within error limits, were indistinguishable from those of the WT (Figure C


Article

Inorganic Chemistry Table 1. EXAFS Simulation Parameters for Wild-Type XDHRc at pH 7.0a XAS N (per Mo) b

X-ray crystallography 2σ2 × 103 (Å2)

R (Å)

b

R (Å)

MoO

1

1.68

2

Mo−O1

Mo−O− Mo−OH MoS

0.59c 0.41c 1b

1.78 1.90 2.18

2b 2b 2b

Mo−O2

Mo−S(MPT)

1b

2.41

2b

Mo−S1(MPT)

Mo−S(MPT)

1b

2.51

2b

Mo−S2(MPT)

Mo···O(E730) Mo···C(MPT)

1b 2b

2.69 3.38

2b 5b

Mo···O(Glu) Mo···C(MPT)

MoS3

1.74(0.07)b 1.88(0.28)c 2.02(0.07)b 2.10(0.41)c 2.19(0.11)b 1.81(0.15)c 2.41(0.06)b 2.51(0.06)c 2.43(0.11)b 2.33(0.12)c 3.32(0.10)b,c 3.56(0.44)b,c

a N, coordination number; R, interatomic distance; 2σ2, Debye−Waller factor. The fit error sum, Rf (calculated for reduced distances of 1.0−3.5 Å), was 6.6% for the given parameters. bValues that were fixed in the fit. cCoordination numbers that were coupled to yield a sum of 1. Data for stepwise improvement of the fit approach are listed in Table S2. Crystallographic values correspond to average distances (standard deviation in parentheses) for structures of XDH and AOX enzymes with a similar Moco in the Protein Data Bank (Table S1 provides further information on the considered structures, ligand annotation is as in Figure 1), in which ban apical MoO group (11 structures)26,28−33 or can apical MoS group (3 structures)49,51 was assigned.

2A). The E730A variant was inactive [activity being ≤0.1% of that of the WT (Figure S3)] in the pH range of 5.5−10.0. The apparent Vmax and KM values for WT and Q179A are plotted versus pH in Figure 2B. For both variants, Vmaxapp increased from pH 5.5 by a factor of ∼20 for increasing the pH to 8.0 and at higher pH values decreased by ∼50% at pH 10.0, whereas KMapp was about constant between pH 5.5−8.0 and ∼10-fold increased up to pH 10.0. In the frame of enzyme kinetics theory, the behavior of Vmaxapp at acidic pH was typical for noncompetitive inhibition whereas the KMapp behavior at alkaline pH suggested competitive inhibition.78 However, the decrease in Vmaxapp at the highest pH values suggested that the substrate and inhibitor concentrations were related, which was explained if the inhibitor was deprotonated xanthine, coupled to xanthine via the Henderson−Hasselbalch equation.79,80 Two equations, which accounted for the reactions shown in the scheme in Figure 3C, were used for the calculation of enzyme kinetics data in a pH range of 5−10, including the same KM (0.058 mM) and Vmax (115 s−1) values and an enzyme protonation (pK1 = 6.8, noncompetitive inhibition, eq 4) or xanthine deprotonation (pK2 = 8.8, competitive inhibition, eq 5) (KI, equilibrium constant of inhibitor binding; Stot, total concentration summing protonated and deprotonated xanthine). V=

V=

V=

Vmax[Stot ] KM + [Stot ]

XAS Analysis of the pH Titration of the WT. The effect of pH variation on the Moco structure in the WT was characterized by XAS. The Mo XANES spectra revealed similar K-edge shapes and energies of 20014.7 ± 0.2 eV in the pH range of 5−10 (Figure 3A), which indicated the presence of Mo(VI) in a distorted square-pyramidal ligand field.63,81 A systematic decrease at more acidic pHs of the amplitude of the pronounced pre-edge absorption feature due to 1s → 4d electronic excitation transitions was described by a titration curve with a pK of 6.8 ± 0.1 (Figure 3A, inset). Because the magnitude of the pre-edge absorption decreases for enhanced centro-symmetry at the metal,82 this observation suggested bond elongation, i.e., of short MoO/S bonds, due to protonation changes at the cofactor at acidic pH. EXAFS analysis provided precise metal−ligand bond lengths at the Moco (Figure 3B).83 First, an EXAFS simulation approach was developed, which described all significant features of the spectrum of the WT at pH 7.0 (Table 1 and Table S2). The simulations revealed the expected two Mo−S(MPT) bonds (∼2.4 and ∼2.5 Å) and respective Mo···C(MPT) distances (∼3.4 Å), complete sulfuration of the cofactor as judged from detection of about one MoS bond of ∼2.2 Å per Mo ion,84 and one short MoO bond (∼1.7 Å). Further oxygen species of molybdenum were attributed to Mo−O− (∼1.8 Å) and Mo−OH (∼1.9 Å) bonds,61 which were present in proportions of ∼0.6 and ∼0.4 per Mo ion, respectively, at pH 7. The latter result suggested that protonation of one oxo ligand at molybdenum resulted in formation of a hydroxo group at acidic pH.61 We note that the formal annotation of the ∼1.8 Å distance to a Mo−O− group is meant to account for the longer distance compared to that of a MoO group, which likely is due to the (gradually) increased negative charge at the oxygen atom and the decreased metal−ligand bond order. The distance difference between the two MoO/O− bonds from the EXAFS fit approach represents the upper limit for the used Debye−Waller parameter. A molybdenum−ligand distance of ∼2.7 Å was attributed to an O atom of the carboxylate group of Glu730. This fit approach provided an excellent description of the experimental data (error sum well below 10%). The EXAFS distances comply considerably better with bond lengths in

(3)

Vmax[Stot ] (KM + [Stot ])(1 + 10 pK1− pH)

(4)

Vmax[Stot ](1 + 10 pK 2 − pH)−1 ⎡ [S ](1 + 10 pH − pK2)−1 ⎤ pK 2 − pH −1 KM⎢⎣1 + tot ) ⎥⎦ + [Stot ](1 + 10 KI (5) app

app

The calculated Vmax and KM values (from Figure S4) well described the pH dependence of the experimental data (Figure 2B). The estimated KI of ∼1.5 mM for the competitive inhibition suggested ∼20-fold weaker binding of deprotonated versus protonated xanthine to the protein. D


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Inorganic Chemistry

was protonated to become a hydroxo at acidic pH. Close to one MoS bond was observed regardless of the pH (Figure 4A), which was shortened from ∼2.19 Å at pH 10.0 to ∼2.16 Å at pH 5.0 (Figure 4B). This small bond length shortening at acidic pH cannot be related to a protonation change at the sulfido ligand, which rather would result in an ∼0.2 Å longer Mo−SH bond.85,86 A slight elongation (∼0.05 Å) at alkaline pH was observed also for the Mo−S(MPT) bonds, whereas the molybdenum−oxygen bond lengths [MoO, Mo−O−, and Mo−O(E730)] showed minor but reproducible changes (≤0.03 Å) over the pH range. The Debye−Waller factor (2σ2) of the Mo−S(MPT) bonds slightly decreased at alkaline pH, suggesting a smaller length difference between the two Mo−S(MPT) bonds compared to that at acidic pH (Figure S6). All bond length changes were described by the same pK of ∼6.8, which was similar to the pK of the decrease in activity at acidic pH, indicating that oxo group protonation to become a Mo−OH group was responsible for the low-pH XDHRc inhibition. The pK of ∼8.8, accounting for inhibition at alkaline pH, had no precedent in the XAS data. DFT Calculations on Moco Models. DFT was employed to further characterize the observed pH-dependent bond length changes at the Moco (Table 2). Model structures were calculated, in which an equatorial (or, for comparison of bond lengths, apical) sulfido ligand was bound at Mo and the oxygen ligands (O1 and O2 in Figure 1) were either present as an OH group or unprotonated (Figure S1). For the unprotonated models, an equatorial sulfido group reproduced the absolute Mo−S(MPT) bond lengths and their bond length difference from XAS consistently, confirming most previous assignments for XDH enzymes.1,9−11,23,24,26−33,37,45,47−50,87 Slight overestimation of Mo−S(MPT) and MoS bond lengths stems from systematic effects in the DFT approach.88,89 The Mo−S1(MPT) bond was generally longer than the Mo− S2(MPT) bond. It is noteworthy that in the Cambridge Structural Database90 and the literature there are no synthetic compounds with systematic MoO/S position variation;85,91−95 however, unprotonated five-coordinated complexes show equatorial MoS bonds (∼2.19 Å) that are longer than apical MoS bonds (∼2.16 Å), and the latter bonds may be further shortened (∼2.13 Å) by an opposite sixth ligand (i.e., Glu730 in XDHRc).95−101 The MoS bond length from XAS (∼2.20 Å) therefore supports an equatorial sulfido group. The experimental asymmetry in the two molybdenum− oxo bond lengths was reproduced only for an apical oxo, with the equatorial O2 atom showing a slightly longer bond. The protonated models well reproduced the experimental MoS bond shortening at low pH. For O1 protonation, an apical hydroxyl yielded two similar Mo−S(MPT) bond lengths, whereas an equatorial O1H yielded a very large Mo−S(MPT) bond length difference, both results being at odds with the experiment. For O2 protonation, an apical oxo group yielded a rather asymmetric Mo−S(MPT) bond length change, with the Mo−S2(MPT) bond length remaining approximately constant and the Mo−S1(MPT) bond now becoming the shorter bond, and a diminished Mo−S(MPT) bond length difference. This finding agreed well with the experimental Mo−S(MPT) bond length spread changes (Figure S6). The Mo−OH bond lengths from DFT and experiment suggested a hydroxo (and not a water) ligand at Mo at acidic pH (Table 2). In summary, the calculations preferentially assigned the equatorial oxygen (O2) to the apparent Mo−O− interaction, being the site of protonation at acidic pH.

Figure 4. pH dependence of Mo coordination for WT and Q179A from EXAFS analysis. (A) pH dependence of coordination numbers (N) of Mo−OH (left y-axis) and MoS (right y-axis) bonds for WT (filled symbols) and Q179A (empty symbols). The solid line shows a fit curve to the N(Mo−OH) values with a pK of 6.8, which is common to both variants; the dashed line emphasizes that N(MoS) is close to unity and pH-independent. (B) pH dependence of the indicated interatomic distances for WT (filled symbols) and Q179A (empty symbols) (underlying experimental and calculated EXAFS data shown in Figure S5). Lines show respective fit curves with a common pK of 6.8. The pH dependence of the Debye−Waller factor of the Mo− S(MPT) bonds is shown in Figure S6.

XDH structures with an apical MoO group instead of an apical MoS group, although the crystal data exhibit comparably large bond length variations (Table 1). Variation of the pH caused pronounced changes in the EXAFS spectra of WT such as an increase in the first Fouriertransform (FT) peak due to MoO bonds and a decrease in the second FT feature with contributions from MoS and Mo−OH bonds at alkaline pH (Figure 3C). The simulation approach described above was used to analyze 20 EXAFS spectra from three independent WT preparations at pH values ranging between 5.0 and 10.0 (Figure S5), and the fit results are compiled in Figure 4. An increase in the number of Mo−OH bonds at the expense of Mo−O− bonds per molybdenum ion from close to zero at pH 10.0 to close to unity at pH 5.0 was observed. This behavior was well described by a titration curve with a pK of 6.8 ± 0.1 (Figure 4A). Accordingly, one oxo ligand E


Article

Inorganic Chemistry Table 2. Molybdenum−Ligand Bond Lengths from DFT for Moco Structure Variationa ΔR (Å)

distance R (Å) exp. DFT exp. DFT

exp. DFT

WT (pH 9) O1apical S3apical WT (pH 5) O1apical, O2H S3apical, O2H O1Hapical S3apical, O1H

S1

S2

2.54 2.62 2.73 2.39 2.46 2.50 2.57 2.65

2.43 2.56 2.60 2.50 2.58 2.58 2.58 2.47

WT (pH 5−9) (O1apical, O2H) − (O1apical) (S3apical, O2H) − (S3apical) (O1Hapical) − (O1apical) (S3apical, O1H) − (S3apical)

ΔR (Å)

distance R (Å)

S1−S2 0.11 0.06 0.13 −0.11 −0.12 −0.08 −0.01 0.18 ΔR (Å)

S3

O1

O2

2.19 2.27 2.26 2.16 2.21 2.22 2.22 2.20

1.69 1.71 1.74 1.69 1.70 1.72 1.99 1.95

1.78 1.75 1.73 1.94 1.89 1.95 1.72 1.71 ΔR (Å)

O1−O2 −0.09 −0.04 0.01 −0.25 −0.19 −0.23 0.27 0.24

S1

S2

S3

O1

O2

−0.15 −0.16 −0.23 −0.05 −0.08

0.07 0.02 −0.02 0.02 −0.13

−0.03 −0.06 −0.04 −0.05 −0.06

0.00 −0.01 −0.02 0.28 0.21

0.16 0.14 0.22 −0.03 −0.02

a

For annotations of O/S ligands at Mo, see Figure 1. In the experimental data (exp.), the longer Mo−S(MPT) bond was denoted S1 at pH 9 or S2 at pH 5 and the shorter MoO bond was denoted O1 at both pH values. H (e.g., in O1H) denotes protonation at the respective oxo group to form an OH ligand. DFT indicates calculated data.

XAS on XDH Variants. Samples of XDHRc variants in the pH range of 5.5−9.5 were characterized by XAS. Compared to WT, Q179A revealed similar shapes of both the XANES and EXAFS spectra and similar spectral changes as a function of pH (Figure 5). Similar K-edge energies (Figures 3 and 5), similar molybdenum−ligand bond lengths (Table 3), and within error limits a similar pK of ∼6.8 (Figures 4 and 5) for the spectral changes were obtained for Q179A and WT. The lack of the Gln179 amino group thus had no significant effect on the geometry and protonation behavior of the Mo ligands. The E730A variant compared to WT showed an ∼0.6 eV higher Kedge energy, and the EXAFS revealed approximately two short MoO bonds, one longer Mo−O bond (∼2.0 Å), and two Mo−S(MPT) bonds. A short MoS bond was barely detectable (Table 3). This result indicated an overall intact Moco structure with a Mo(VI) ion in E730A, implied that the sulfido was replaced with an oxo ligand, and suggested that one OH group was bound at Mo. The coordination number (N) of a long Mo−O distance was very small, so that such a distance was practically undetectable in E730A. This finding supports the suggestion that the carboxyl group of Glu730 accounted for the ∼2.7 Å Mo−O distance in the WT and that in the absence of Glu730, the putative void was not occupied, for example, by a water molecule at a similar distance from Mo. The XAS spectra of E730A within noise limits were pH invariant, meaning that low-pH protonation of the altered metal site did not occur in the presence of an OH ligand already at alkaline pH.

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DISCUSSION Moco Structure in XDHRc. Our XAS data, in agreement with previous XAS results,61,62 unambiguously establish that, besides the two Mo−S(MPT) bonds, the Mo(VI) ion in oxidized WT XDHRc shows two short molybdenum−oxygen bonds at pH ≥8.0 and one short molybdenum−sulfur bond in the pH range of 5−10. A Mo−O− interaction, considered as a deprotonated hydroxo (OH) ligand, is discriminated by its bond being ∼0.05 Å longer than the MoO bond, as discussed previously.61 An approximately constant and

Figure 5. XAS data for XDHRc variants at two pH values. (A) XANES spectra (vertically displaced). The inset shows bond valence sums (BVS, calculated with eq 2 from the EXAFS fit parameters in Table 3) plotted vs K-edge energies (at the 50% level). (B) FTs of EXAFS spectra in panel C (black lines, experimental data; colored lines, fits with parameters listed in Table 3).

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Inorganic Chemistry Table 3. EXAFS Simulation Parameters for XDHRc Variantsa N (per Mo), R (Å), 2σ2 × 103 (Å2) variant WT WT Q179A Q179A E730A E730A

pH 6 9 6 9 6 9

MoO b

1.3, 1.69, 8 1.9,b 1.72, 7 1.4,b 1.68, 8 1.9,b 1.73, 9 1.7, 1.72, 8c 1.8, 1.73, 8c

Mo−OH b

MoS c

0.7, 1.94, 2 0.1,b 1.98, 2c 0.6,b 1.91, 2c 0.1,b 1.90, 2c 1.0, 2.03, 2c 1.1, 2.04, 2c

1.0, 0.9, 1.1, 0.9, 0.1, 0.2,

2.16, 2.19, 2.15, 2.19, 2.10, 2.12,

Mo−S(MPT) c

2 2c 2c 2c 2c 2c

c

2, 2.43, 10 2,c 2.49, 9 1.9, 2.44, 11 2.0, 2.48, 10 2.1, 2.40, 8 2.1, 2.41, 6

Rf (%)

Mo···O 1.2, 0.9, 1.2, 1.0, 0.3, 0.2,

2.63, 2.64, 2.62, 2.65, 2.61, 2.67,

c

5 5c 5c 5c 5c 5c

6.7 5.5 8.7 5.2 5.8 7.3

a

Data correspond to EXAFS spectra in Figure 5. bParameters that were coupled to yield a sum of 2; for further annotations, see Table 1. cParameters that were fixed in the simulations.

Figure 6. Molybdenum site structures in XDHRc. The global Moco geometry follows the crystal structure.28 Indicated bond lengths (in angstroms, rounded in the second digit) at pH 9 or 5 were here determined by EXAFS. Relative orientations of O/S ligands at Mo are suggested by our XAS and DFT results and by crystal structures. The orientation of the Glu730 carboxylic side chain is tentative. The pK of ∼6.8 for Mo−O− protonation to form a Mo−OH group accounts for enzyme inhibition at acidic pH. Assignment of the Mo−O− group to the equatorial oxygen is favored by our data.

relatively short molybdenum−Glu730 distance implied that the carboxyl remains unprotonated between pH 5 and 10 and forms a weak bond with the metal. Comparison to bond lengths in synthetic compounds assigns the short molybdenum−sulfur bond to a MoS group as opposed to a Mo−SH group.85,86,96,102,103 The MoS group was found in stoichiometric amounts and at all pH values in functional XDHRc, meaning that static protonation of the sulfido ligand without substrate did not occur. In crystal structures with an equatorial sulfido ligand, on average the equatorial MoO2 bond (∼2.0 Å) was considerably longer than the apical MoO1 bond (1.7 Å), which has been taken as evidence of the presence of a hydroxyl group. The XAS and DFT data favored assignment of O2 to the Mo−O− group, which, however, shows a much shorter bond length (∼1.75 Å) than in many crystal structures. The longer bond in the diffraction data may be related to resolution limitations, to mixtures of unprotonated and protonated Mo sites in the crystals, and/or to reduction of initial Mo(VI) sites and accompanying O2 protonation in the X-ray beam.104−106 Our XAS data exclude a Mo−OH group in functional XDHRc at alkaline pH (Figure 6). Interestingly, the DFT data suggested that the Mo−S1(MPT) bond is shorter than the Mo− S2(MPT) bond in the unprotonated site and that this is reversed in the site protonated at O2. The Debye−Waller factor of the Mo−S(MPT) bonds from EXAFS supports such bond lengths. These findings imply a considerable change in the geometry of the pterin ligand upon protonation of the site. A sulfido ligand was absent in the E730A variant, where it was replaced with a MoO group, and the metal further carried one oxo and one hydroxyl ligand, regardless of pH. So

Figure 7. Mechanistic proposal for xanthine to uric acid conversion in XDH. A Mo(VI) ion with equatorial sulfido and initially deprotonated MoO/O− ligands (A) is required for the first electron and proton transfer steps leading to a Mo(IV) intermediate with a bound substrate (B), followed by two-electron abstraction to regain Mo(VI) (C) during bond hydrolysis involving Glu730 as a putative proton shuttle and oxygen atom transfer to the product. The Mo−SH proton may be used for product reprotonation or released (dashed arrow), and more elaborate protonation sequences are conceivable (see, e.g., ref 23). We note that further and so far unknown intermediates may be expected to occur during the electron, proton, and group transfer reactions, which may be addressed in future studies. For further details, see the text.

far, this variant has been thought to be inactive because of the missing glutamate, which was consequently proposed to function as an active site base.1,10,11,23,27 We show here that the variant does not contain the sulfido ligand, which is essential for enzyme activity. The presence of a hydroxyl group already at alkaline pH further contributes to the inability of the enzyme to react with xanthine. A possible reason for the presence of the desulfo Moco in the E730A variant may be G


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Inorganic Chemistry

reaction,9,116,117 facilitating both metal reduction steps at a similar redox potentials with a single metal site protonation. Mechanistic Considerations. A “catalytically labile” hydroxyl group and the sulfido ligand as a proton acceptor have been involved in mechanistic proposals for xanthine oxidation in XDH.1,10,11,23,27,28,45,48 MoS group protonation was evidenced by bond elongation in crystal structures with the substrate bound at Mo.48,118 Our data rule out the possibility that an initially present hydroxyl at Mo(VI) is relevant in catalysis. However, such a group may be created by proton transfer from xanthine to, i.e., the equatorial Mo−O− group. The MoS group then may accept a proton in later reaction stages. Alternatively, initial MoS protonation may render a hydroxyl function in substrate binding to the metal obsolete. The second option seems preferable as it provides a simplified reaction path. In both scenarios, the sulfido ligand may not be critical for determining the redox potential of the Mo site109,110 but may shift the pK of the Mo−O− group toward acidic pH, so that it remains unprotonated under physiological conditions.119 We summarize our attributions in Figure 7. Two-electron xanthine oxidation at Mo(VI) may involve proton transfer to the MoS group and nucleophilic attack of the Mo−O− group at C8 to form Mo(IV) with a bound substrate, followed by 2e− oxidation of Mo(IV), proton transfer to the product during O atom transfer in a hydrolysis step, and proton release possibly via Glu730, leading to uric acid. We emphasize that formation of the conjugated base urate (pK ∼ 5.4)120 at alkaline pH may imply different proton transfer/release sequences and that such schemes do not exclude more intricate (e.g., interligand) proton movements.23 In any event, a deprotonated Moco structure seems to be a crucial factor for catalysis in the XDH family.

unspecific insertion of this cofactor form due to the usage of the heterologous expression system in E. coli. Another option is that Glu730 is involved in the insertion of sulfurated Moco into the enzyme and/or in the Moco sulfuration step itself.38−40 Overall, the specific metal coordination geometry with a sulfido ligand and the presence of Glu730 seem to stabilize an unprotonated metal site in WT XDHRc. Enzyme Inhibition and Protolytic Events. Our present and previous61,62 XAS studies have revealed pH-dependent bond length changes at the Moco in WT XDHRc, which are assigned to a Mo−O− protonation that forms a Mo−OH ligand (Figure 6). The pK of ∼6.8 of Mo−OH bond formation was identical to the pK of the activity decrease at acidic pH. The low-pH inhibition of xanthine oxidation activity in XDHRc thus is directly caused by Mo−OH bond formation at the active site. Crystal structures suggest at least a weak hydrogen bond between the amino group of Gln179 and the apical Mo ligand, which is expected to significantly influence the protonation behavior of the apical MoO group. As both WT and Q179A revealed similar activity and protonation behavior and the pK of the equatorial oxygen may be much less affected by the Q179A exchange, the equatorial oxygen is favored as the site of protonation. Enzyme kinetics revealed a second pK of ∼8.8 for inhibition at alkaline pH due to a deprotonation, which did not alter the bond lengths at Mo and likely does not occur at the Moco itself. A relation of the pK to the amino acids close to the Moco (Gln179, Glu730, and Glu232), to redox events of the flavin or iron−sulfur cluster cofactors of XDHRc, or to NAD+ reduction is unlikely.41,43,107−111 The pK of xanthine in aqueous solution is ∼7.7,112,113 but the pK of enzyme-bound xanthine likely is increased by at least 1 unit.43 Therefore, the pK of ∼8.8 is attributed to the formation of deprotonated xanthine, acting as a competitive inhibitor. Because deprotonated xanthine is easier to oxidize,114 the inactivation may not be related to the redox properties of the two substrate forms. Xanthine binding at the Mo site requires deprotonation at its C8 atom, which becomes less acidic after N3 deprotonation.112 Impaired proton transfer from deprotonated xanthine thus may prevent its oxidation. Mo−OH bond formation inhibits XDHRc in a noncompetitive fashion; i.e., it may not hinder binding of xanthine to the protein. Electrochemistry has provided redox midpoint potentials (Em) for Mo(VI) → Mo(V) → Mo(IV) reduction in XDH enzymes in the absence of substrate.109,110,115 The Em of the Mo(V)/Mo(VI) pair is constant below pH ∼7.5 and decreases above pH ∼8 (−450 mV at pH ∼8.5), whereas the Em of the Mo(IV)/Mo(V) pair increases below pH ∼7.5 and is constant above pH ∼8 (−450 mV at pH ∼8.5).110 In contrast, desulfo XDH shows similar Em values below pH ∼8 but a continued Em decrease above pH ∼8 for Mo(IV)/Mo(V) reduction.110 Combining these and our results with Nernst theory suggests that for the sulfurated site, at pH < pK (∼6.8) reduction of the Mo(VI)−OH is not coupled and of Mo(V)− OH is coupled to a protonation, but at pH > pK, reduction of the Mo(VI)−O− is coupled and of Mo(V)−OH is not coupled to a protonation. Both reduction steps require a protonation at the desulfo site. XDHRc inhibition at acidic pH or in the absence of a sulfido ligand hence may not reflect the Mo redox properties, predicting easier reduction of a Mo(VI)−OH site. It may rather be due to the lack of an acceptor for the xanthine proton because Mo site protonation is impaired for an initial Mo−OH group. These considerations support the view that Mo(VI) reduction is a proton-coupled electron transfer

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02846. Crystal structures of XDH and AOX enzymes (Table S1), Moco model structures from DFT (Figure S1), enzyme kinetic data (Figure S2), activity of XDHRc variants (Figure S3), calculated enzyme kinetics (Figure S4), EXAFS data of WT and Q179A (Figure S5), EXAFS simulation refinement (Table S2), and Debye−Waller factors of Mo−S(MPT) bonds (Figure S6) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany. Phone: +49 30 838 56101. E-mail: [email protected]. *Institut für Biochemie and Biologie, Molekulare Enzymologie, Universität Potsdam, Karl-Liebknecht Strasse 24-25, 14476 Potsdam, Germany. Phone: +49 331 977 5603. E-mail: sleim@ uni-potsdam.de. ORCID

Michael Haumann: 0000-0001-7008-1764 Present Address §

K.G.V.S.-C.: MAX IV Laboratory, Lund University, 22100 Lund, Sweden. H


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Inorganic Chemistry Notes

(17) Kikuchi, H.; Fujisaki, H.; Furuta, T.; Okamoto, K.; Leimkühler, S.; Nishino, T. Different inhibitory potency of febuxostat towards mammalian and bacterial xanthine oxidoreductases: insight from molecular dynamics. Sci. Rep. 2012, 2 (331), 1−8. (18) Tada, Y.; Suzuki, J. Oxidative stress and myocarditis. Curr. Pharm. Des. 2016, 22, 450−471. (19) Battelli, M. G.; Polito, L.; Bortolotti, M.; Bolognesi, A. Xanthine oxidoreductase in drug metabolism: beyond a role as a detoxifying enzyme. Curr. Med. Chem. 2016, 23, 4027−4036. (20) Panth, N.; Paudel, K. R.; Parajuli, K. Reactive oxygen species: a key hallmark of cardiovascular disease. Adv. Med. 2016, 2016, 9152732. (21) Borges, F.; Fernandes, E.; Roleira, F. Progress towards the discovery of xanthine oxidase inhibitors. Curr. Med. Chem. 2002, 9, 195−217. (22) Wang, C. H.; Zhang, C.; Xing, X. H. Xanthine dehydrogenase: An old enzyme with new knowledge and prospects. Bioengineered 2016, 7, 395. (23) Hille, R.; Hall, J.; Basu, P. The mononuclear molybdenum enzymes. Chem. Rev. 2014, 114, 3963−4038. (24) Romao, M. J. Molybdenum and tungsten enzymes: a crystallographic and mechanistic overview. Dalton Trans. 2009, 4053−4068. (25) Brondino, C. D.; Romao, M. J.; Moura, I.; Moura, J. J. Molybdenum and tungsten enzymes: the xanthine oxidase family. Curr. Opin. Chem. Biol. 2006, 10, 109−114. (26) Cao, H.; Hall, J.; Hille, R. X-ray crystal structure of arseniteinhibited xanthine oxidase: mu-sulfido,mu-oxo double bridge between molybdenum and arsenic in the active site. J. Am. Chem. Soc. 2011, 133, 12414−12417. (27) Huber, R.; Hof, P.; Duarte, R. O.; Moura, J. J.; Moura, I.; Liu, M. Y.; LeGall, J.; Hille, R.; Archer, M.; Romao, M. J. A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 8846−8851. (28) Dietzel, U.; Kuper, J.; Doebbler, J. A.; Schulte, A.; Truglio, J. J.; Leimkühler, S.; Kisker, C. Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase. J. Biol. Chem. 2009, 284, 8768−8776. (29) Ishikita, H.; Eger, B. T.; Okamoto, K.; Nishino, T.; Pai, E. F. Protein conformational gating of enzymatic activity in xanthine oxidoreductase. J. Am. Chem. Soc. 2012, 134, 999−1009. (30) Cao, H.; Pauff, J. M.; Hille, R. Substrate orientation and catalytic specificity in the action of xanthine oxidase: the sequential hydroxylation of hypoxanthine to uric acid. J. Biol. Chem. 2010, 285, 28044−28053. (31) Correia, M. A.; Otrelo-Cardoso, A. R.; Schwuchow, V.; Sigfridsson Clauss, K. G.; Haumann, M.; Romao, M. J.; Leimkühler, S.; Santos-Silva, T. The Escherichia coli periplasmic aldehyde oxidoreductase is an exceptional member of the xanthine oxidase family of molybdoenzymes. ACS Chem. Biol. 2016, 11, 2923−2935. (32) Coelho, C.; Foti, A.; Hartmann, T.; Santos-Silva, T.; Leimkühler, S.; Romao, M. J. Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase. Nat. Chem. Biol. 2015, 11, 779−783. (33) Coelho, C.; Mahro, M.; Trincao, J.; Carvalho, A. T.; Ramos, M. J.; Terao, M.; Garattini, E.; Leimkuhler, S.; Romao, M. J. The first mammalian aldehyde oxidase crystal structure: insights into substrate specificity. J. Biol. Chem. 2012, 287, 40690−40702. (34) Ilich, P.; Hille, R. Oxo, sulfido, and tellurido Mo-enedithiolate models for xanthine oxidase: understanding the basis of enzyme reactivity. J. Am. Chem. Soc. 2002, 124, 6796−6797. (35) Cerqueira, N. M.; Fernandes, P. A.; Gonzalez, P. J.; Moura, J. J.; Ramos, M. J. The sulfur shift: an activation mechanism for periplasmic nitrate reductase and formate dehydrogenase. Inorg. Chem. 2013, 52, 10766−10772. (36) Schrapers, P.; Hartmann, T.; Kositzki, R.; Dau, H.; Reschke, S.; Schulzke, C.; Leimkühler, S.; Haumann, M. Sulfido and cysteine ligation changes at the molybdenum cofactor during substrate conversion by formate dehydrogenase (FDH) from Rhodobacter capsulatus. Inorg. Chem. 2015, 54, 3260−3271.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) within the Berlin Cluster of Excellence “Unifying Concepts in Catalysis” (EXC 314). M.H. thanks the Bundesministerium für Bildung und Forschung for funding (Grant 05K14KE1). S.L. thanks the DFG for financial support (Grants LE1171/6-2 and LE1171/11-1). K.G.V.S.-C. thanks the Swedish “Bengt Lundqvist Minne” and the WennerGren Foundation for fellowships. We thank V. Briois and E. Fonda at the SAMBA beamline of SOLEIL for excellent technical support and J. Kurtzke (Universität Potsdam) for protein purification.

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ABBREVIATIONS AOX, aldehyde oxidase; BVS, bond valence sum; DFT, density functional theory; EXAFS, extended X-ray absorption fine structure; MPT, molybdopterin; Moco, molybdenum cofactor; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spectroscopy; XDH, xanthine dehydrogenase

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