Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

Background: Copper amine oxidases catalyze amine oxidation using copper and a quinone cofactor.

Results: Halides bind axially to the copper center, preventing the reduced cofactor from adopting an on-copper conformation.

Conclusion: The cofactor undergoes large conformational changes during the catalytic reaction that enable transitions between different types of chemistry.

Significance: Molecular details of cofactor movement have been unveiled based on structural and kinetic evidence.

Abstract

The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQ_amr) that is in equilibrium with a semiquinone radical (TPQ_sq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQ_sq in an “on-copper” conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the “off-copper” conformation of TPQ_amr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQ_amr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQ_amr/TPQ_sq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.

Introduction

Copper amine oxidases (CAOs²; EC 1.4.3.6) catalyze the oxidative deamination of various primary amines to produce the corresponding aldehydes and ammonia, coupled with the reduction of molecular oxygen to hydrogen peroxide (1–3). CAOs play distinct physiological roles in prokaryotes and eukaryotes. Prokaryotic CAOs mainly function to assimilate primary amines as carbon and nitrogen sources for growth. Eukaryotic CAOs have versatile functions, being involved in the detoxification of bio-active amines, such as histamine (4); cell adhesion (5); cell death (6); collagen cross-linking in animals (7); and germination, root growth, and healing of wounded cell walls in plants (8). It has been reported that human serum CAOs cause angiopathy in diabetes (9). Therefore, various CAO inhibitors have been developed as therapeutic drugs (10, 11).

The catalytic center common to the CAO family consists of a mononuclear copper ion and a redox-active organic cofactor, topa quinone (TPQ), which is generated through posttranslational and self-catalytic processes (12–15). As summarized in a recent review (3), x-ray crystal structures of CAOs have been determined for a number of enzymes from various sources, including bacteria (Escherichia coli (16) and Arthrobacter globiformis (AGAO) (17–19)), yeasts (Hansenula polymorpha (recently reclassified as Pichia angusta) (HPAO-1 and HPAO-2) (20, 21) and Pichia pastoris (lysyl oxidase) (22)), a fungus (Aspergillus nidulans (23)), a plant (Pisum savitum (24)), a mammal (Bos taurus (25)), and Homo sapiens (diamine oxidase and vascular adhesion protein 1, which is identical to semicarbazide-sensitive amine oxidase (26–28)). The active site structures of these enzymes, including the positions of TPQ and Cu(II), are highly conserved, suggesting that they have a common mechanism for single-turnover TPQ biogenesis and catalytic amine oxidation. The latter process has been shown to proceed through a ping-pong mechanism consisting of two half-reactions (Scheme 1) (1–3).

SCHEME 1.

Presumed catalytic mechanism of AGAO.

In the initial reductive half-reaction, the C5 carbonyl group of the oxidized cofactor (TPQ_ox) undergoes nucleophilic attack by amine substrate to form the substrate Schiff base (TPQ_ssb). Stereospecific proton abstraction by a conserved base (Asp-298 in AGAO) (29) converts TPQ_ssb to the product Schiff base (TPQ_psb). Concomitantly with the release of the corresponding aldehyde, TPQ_psb is hydrolyzed to the reduced cofactor (an aminoresorcinol form, TPQ_amr) that is in equilibrium with a semiquinone radical form (TPQ_sq) via the single electron transfer reduction of Cu(II) to Cu(I). In these steps of the reductive half-reaction, the cofactor ring is not directly ligated to the copper atom (a configuration designated the “off-copper” conformation), whereas in the TPQ_sq·Cu(I) state, the cofactor ring is assumed to be directed toward the metal in the so-called “on-copper” conformation to facilitate electron transfer. This suggests that the equilibrium between TPQ_amr·Cu(II) and TPQ_sq·Cu(I) is accompanied by a substantial conformational reorganization of the TPQ ring. Indeed, the conformational flexibility of TPQ within the active site of CAOs has previously been suggested to be important in catalysis (24, 30, 31). In the subsequent oxidative half-reaction, the reduced cofactor is reoxidized by dioxygen to produce hydrogen peroxide and an iminoquinone intermediate (TPQ_imq), which is further hydrolyzed to form the oxidized cofactor, releasing ammonia in the following step (1–3). In the presence of excess substrate, it is assumed that TPQ_imq reacts directly with the substrate amine to form TPQ_ssb (via a trans-imination reaction) rather than regenerating TPQ_ox (Scheme 1).

Depending on the enzyme sources and reaction conditions, such as pH and temperature, various amounts of TPQ_sq·Cu(I) are known to be formed by adding an amine substrate under anaerobic conditions, which is assumed to induce the reductive half-reaction. However, the mechanistic role of the TPQ_sq·Cu(I) form in the subsequent O₂ reduction remains unclear and controversial (32–39). Two reaction pathways for the O₂ reduction have been proposed, depending on the enzyme sources. One is an inner sphere mechanism in which O₂ is coordinated on Cu(I) and reduced by the transfer of two electrons from TPQ_sq·Cu(I) to ultimately produce a Cu(II)-bound peroxide species and TPQ_imq (Scheme 1). The necessary singlet to triplet spin transition is allowed by the coordination of O₂ to Cu(I). AGAO and P. savitum CAO are CAOs that have been suggested to follow this mechanism (37, 38). The formation of the TPQ_sq·Cu(I) state at the beginning of the oxidative half-reaction is believed to be essential in this process. The other mechanism that has been proposed is an outer sphere process that is suggested to occur in bovine serum CAO (34) and HPAO-1 (36, 39). In both of these enzymes, O₂ binds to a hydrophobic pocket close to the cofactor and is initially reduced by TPQ_amr via a single electron transfer that does not change the oxidation state of the Cu(II) center. The resulting superoxide anion then coordinates to Cu(II), inducing the second electron transfer. The TPQ_sq·Cu(I) complex is not formed in this catalytic cycle and is assumed to be an off-pathway product generated only by the anaerobic reduction of the CAO by the amine substrate.

We have previously determined x-ray crystal structures of the intermediates, including TPQ_ssb and TPQ_psb, formed during the reductive half-reaction of AGAO with 2-phenylethylamine (2-PEA) (29), tyramine (40), and ethylamine (41). In each case, the TPQ cofactor in these structures had an off-copper conformation. Here, we present new high resolution structures of TPQ_sq formed in the reductive half-reaction of AGAO with 2-PEA and histamine. The TPQ_sq formed with these substrates exists exclusively in the on-copper conformation with the 4-OH group ligating axially to the copper center, which is probably in the Cu(I) oxidation state. Moreover, we found that the off-copper TPQ_amr is formed when the crystals are soaked with substrate in the presence of halide ions that act as uncompetitive inhibitors with respect to the amine substrate and noncompetitive inhibitors with respect to dioxygen. Halide ions bind axially to the copper center, preventing TPQ_amr from coordinating to copper. Combined with the results of spectrophotometric, steady-state, and transient kinetics analyses, the results presented herein provide unequivocal evidence for the occurrence of a large conformational change in the TPQ cofactor during the reductive half-reaction of AGAO.

Experimental Procedures

Materials

Recombinant AGAO was purified as its inactive precursor and converted to the copper- and TPQ-containing active form as reported previously (12, 19). Protein and TPQ_sq concentrations were determined spectrophotometrically using molar extinction coefficients of ϵ₂₈₀ = 93,200 m⁻¹ cm⁻¹ (12) and ϵ₄₆₈ = 4500 m⁻¹ cm⁻¹, respectively (35). All amine substrates used for kinetic analyses and crystal soaking were neutralized with 1 m H₂SO₄.

Spectrophotometric Measurements

To achieve fully anaerobic conditions, the enzyme and substrate solutions were kept in a vacuum-type glove box (Iuchi, SGV-65V) filled with 99.999% (v/v) argon gas for at least 2 h, as described previously (18). The enzyme (final concentration, 100 μm monomer) was anaerobically mixed with 1 mm 2-PEA in 50 mm HEPES, pH 6.8, in the presence or absence of various concentrations of sodium, potassium, or ammonium salts of halide ions. For measurements of pH dependence, the enzyme (100 μm monomer) was reduced with substrate in 100 mm MES (pH 5.7, 5.8, 6.0, 6.3, 6.5, and 6.7), 100 mm HEPES (pH 7.0, 7.3, 7.5, 7.8, and 8.0), 100 mm TAPS (pH 8.5 and 9.0), or 100 mm CHES (pH 9.5 and 10.1) at a nearly constant ionic strength (I = 0.35 ± 0.03) after adjustment with 100 mm Na₂SO₄. The enzyme was mixed with its substrate under anaerobic conditions in a quartz cuvette with a gas-tight screw cap, and after 5 min, the absorption spectrum was measured at 25 °C with an Agilent 8453 photodiode array spectrophotometer. An apparent pK_a value for the absorbance change at 468 nm was determined by fitting the data to Equation 1,

where y represents the absorbance at a particular pH, C₁ and C₂ are the pH-independent values of the absorbance, and K_a1 and K_a2 are the acid dissociation constants associated with the pH profile. Data fitting was performed by nonlinear regression using Kaleidagraph version 4.1 (Abelbeck Software).

Steady-state Kinetic Analysis

Steady-state kinetic analyses were conducted at 30 °C with 2-PEA, histamine, or ethylamine (hydrochloride) as the substrate using the colorimetric assay protocol described previously (12). Inhibition by halide ions was studied using assay solutions containing 2–40 μm 2-PEA and 0–50 mm NaF, NaCl, NaBr, or NaI. Inhibition constants (K_i) were determined on the basis of uncompetitive inhibition (42) with respect to the amine substrate using Equation 2,

where v₀ represents the initial reaction rate and [S] and [I] are the concentrations of substrate and inhibitor, respectively. Data fitting was performed by multiple regression analysis using R (available from the R Project Web site). Inhibition by halide ions was also studied using assay solutions containing 8.8–1160 μm dissolved oxygen and a fixed concentration (40 μm) of 2-PEA in the presence of 0–25 mm NaCl. Various concentrations of dissolved oxygen were established by mixing 99.99% (v/v) O₂ and 99.99% (v/v) N₂ gas with a gas mixer (KOFLOC, PMG-1) and bubbling the resulting mixture through a needle into the reaction mixture contained in a tightly sealed cuvette with a silicon rubber cap. The mixed gas line was also branched into the cell of a Clark-type oxygen electrode (YSI Inc., model 5300) for determination of the dissolved oxygen concentration. The reaction was initiated by adding a small amount of the anaerobic enzyme solution with a microsyringe. Inhibition constants (K_i) were determined on the basis of noncompetitive inhibition (42) with respect to dissolved oxygen using Equation 3,

where v₀ represents the initial reaction rate, and [S] and [I] are the concentrations of dissolved oxygen and inhibitor, respectively. Data fitting was performed by multiple regression analysis using R.

The effect of solvent viscosity was investigated by measuring the enzyme's activity toward 2-PEA at 4 °C in a solution of 0–30% (w/v) glycerol or sucrose as a viscogen; the solvent's viscosity was determined relative to a buffer-only solution with an Ostwald viscometer.

Stopped-flow Measurements

Transient kinetic analyses were done at 4 °C with an Applied Photophysics stopped-flow spectrophotometer (40, 41). Typically, equal volumes (about 30 μl each) of enzyme (200 μm monomer in 50 mm HEPES buffer, pH 6.8) and substrate (1 mm 2-PEA) solutions were mixed in a 20-μl mixing cell by triggering with an N₂ gas piston; the mixing dead time was generally 2.3 ms at an N₂ gas pressure of 500 kilopascals. To avoid spectral changes associated with the oxidative half-reaction, both enzyme and substrate solutions were maintained under fully anaerobic conditions as described above. The substrate solution was supplemented with 200 mm NaCl, 600 mm NaBr, or 600 mm Na₂SO₄, as appropriate. The effect of solvent viscosity on transient kinetics was also studied as described above by adding a viscogen (0–30% (w/v) glycerol or sucrose) to both the enzyme and substrate solutions. UV-visible absorption spectra were recorded every 2.5 ms at wavelengths of 250–800 nm. Spectral data were analyzed using Pro-Kineticist II (Applied Photophysics) to obtain the spectra of the reaction intermediates and to calculate the rate constants for each reaction step.

Single-crystal Microspectrophotometry

AGAO crystals prepared as described below were subjected to microspectrophotometry before x-ray diffraction as reported previously (29).

X-ray Crystallographic Analysis

AGAO was crystallized by microdialysis essentially according to the method described previously (29). Briefly, a 15 mg/ml protein solution was dialyzed in a 50-μl dialysis button at 16 °C against 1.05 m potassium-sodium tartrate in 25 mm HEPES buffer, pH 6.8. After 2 weeks of crystal growth, the dialysis buttons were transferred into a fresh reservoir solution supplemented with 45% (v/v) glycerol as a cryoprotectant, and the crystals were soaked at 16 °C for 24 h, followed by further soaking in the fully anaerobic reservoir solution containing 45% (v/v) glycerol for 24 h. The crystals were then incubated in a solution (pH 6.8) containing 4 mm 2-PEA, 10 mm histamine, or 50 mm ethylamine (hydrochloride) with or without 100 mm NaCl or 300 mm NaBr for about 1 h until their color faded. They were then mounted on thin nylon loops (φ, 0.5–0.7 mm) and frozen by flash cooling in liquid CF₄. All procedures were done in the anaerobic box, and the frozen crystals were kept in liquid N₂ until x-ray diffraction analysis.

Diffraction data sets were collected at 100 K with synchrotron X-radiation using a DIP6040 imaging plate (Bruker AXS, Billerica, MA) in the BL44XU station or using a Quantum 210 CCD detector (ADSC) in the BL38B1 station at SPring-8 (Hyogo, Japan). X-rays with a wavelength of 0.919 Å were used to detect anomalous peaks derived from bromine; a wavelength of 0.9 Å was used otherwise. The collected data sets were processed and scaled using HKL2000 (43) or MOSFLM (44) and SCALA (45), respectively. The starting model was obtained by molecular replacement with Phaser (46). The search model was based on the coordinates of the AGAO monomer (Protein Data Bank code 1IU7) after removing all water molecules and a metal ion. Refinements, electron density map calculations, and assignment of solvent molecules were initially done using Refmac 5 (47) and later with Phenix (48). Manual rebuilding was performed using Coot (49), and water molecules and other ligands, such as metal ions, were added step by step to the model during the refinement process. The models of the catalytic intermediates of TPQ and phenylacetaldehyde were built using the Monomer library sketcher from the CCP4 package (50), and then the dictionary files used with Refmac 5, Phenix, and Coot were generated using the PRODRG server (51). PyMOL version 1.5 (Schrödinger, LLC) was used for figure drawings. Anomalous maps for bromine atoms were generated using fft (52) from the CCP4 package (50) based on anomalous difference and phase data for the final model. Details and statistics pertaining to the data collection and refinement are summarized in Table 1.

TABLE 1.

Data collection and crystallographic refinement statistics

	AGAOPEA	AGAOHTA	AGAOETA/HCl	AGAOPEA/NaBr	AGAOPEA/NaCl	AGAONaBr
Soaking conditions	4 mm 2-PEA, anaerobic	10 mm histamine, anaerobic	50 mm ethylamine-HCl, anaerobic	4 mm 2-PEA, 300 mm NaBr, anaerobic	4 mm 2-PEA, 100 mm NaCl, anaerobic	300 mm NaBr, aerobic
Protein Data Bank code	3X3X	3X3Y	3X3Z	3X41	3X40	3X42
Data collection
Temperature (K)	100	100	100	100	100	100
Wavelength (Å)	0.9	0.9	0.9	0.919	0.9	0.919
Space group	C2	C2	C2	C2	C2	C2
Unit-cell dimensions
a, b, c (Å)	191.66, 62.89, 158.01	193.47, 63.25, 157.91	192.55, 62.73, 157.65	191.62, 63.29, 157.85	191.81, 63.02, 158.11	192.68, 63.51, 158.10
β (degrees)	117.48	117.73	117.62	117.21	117.29	117.55
Resolution limit (Å)	38.5–1.57 (1.65–1.57)	50.0–1.50 (1.53–1.50)	38.5–1.51 (1.59–1.51)	47.3–1.87 (1.97–1.87)	26.5–1.85 (1.95–1.85)	100–1.89 (1.96–1.89)
No. of observations	1,511,400	1,700,730	1,862,064	503,626	520,004	935,217
No. of unique reflections	228,324	264,870	260,515	137,175	142,096	267,101
I/σ (I)a	5.3 (1.9)	26.1 (19.1)	17.3 (4.0)	13.0 (2.6)	10.1 (3.5)	6.6 (1.7)
Redundancya	6.6 (6.2)	6.4 (6.0)	7.1 (6.9)	3.7 (3.5)	3.7 (3.8)	3.5 (3.2)
Overall completeness (%)a	98.1 (96.0)	98.1 (97.4)	99.8 (99.8)	98.7 (97.2)	99.2 (98.8)	99.3 (98.7)
Overall Rmerge (%)a,b	9.3 (39.2)	8.8 (42.5)	9.2 (40.4)	9.1 (35.1)	11.2 (38.2)	5.6 (34.7)
Wilson B factor (Å2)	13.85	13.84	11.87	16.52	16.85	19.81
No. of molecules/asymmetric unit	2	2	2	2	2	2
Refinement statistics
Resolution limit (Å)	20.4–1.57 (1.59–1.57)	36.5–1.50 (1.52–1.50)	22.3–1.51 (1.53–1.51)	37.1–1.87 (1.89–1.87)	26.2–1.85 (1.87–1.85)	24.7–1.89 (1.90–1.88)
Residues in the core φψ region (%)	96.4	96.7	96.3	96.4	96.2	96.5
No. of atoms/asymmetric unit	11,032	11,413	11,677	11,201	11,118	11,191
No. of solvent atoms	1170	1236	1527	1134	1239	1198
Average temperature factors
Protein	18.5	17.5	17.5	21.5	24.0	23.2
Ligand/ion	29.1	35.0	33.2	38.9	37.3	48.2
Solvent	29.2	29.2	31.8	34.7	33.3	34.5
Root mean square deviation from ideal values
Bond lengths (Å)	0.011	0.012	0.011	0.014	0.013	0.009
Bond angles (degrees)	1.049	1.078	1.153	1.112	1.077	1.083
Rwork (%)a,c	21.5 (26.2)	16.2 (22.4)	16.1 (20.3)	15.2 (20.5)	21.2 (28.1)	15.8 (22.6)
Rfree (%)a,d	24.3 (30.5)	17.7 (25.7)	17.8 (21.9)	18.0 (23.7)	26.4 (34.1)	19.0 (27.9)
Ramachandran plot statistics (%)
Residues in favored regions	96.4	96.7	96.3	96.3	96.1	96.6
Residues in allowed regions	3.6	3.3	3.5	3.7	3.7	3.4
Outliers	0	0	0.2	0	0.2	0

^a Values in parentheses refer to data for the highest resolution shells.

^b R_merge = Σ_hΣ_i|I_h,1 − 〈I_h〉|/Σ_hΣi I_h,1, where I_h,1 is the intensity value of the ith measurement of h, and 〈I_h〉 is the corresponding mean value of I_h for all i measurements.

^c R_work = Σ‖F_o| − |F_c‖/Σ|F_o|.

_d R_free is an R factor of the refinement evaluated for 5% of reflections that were excluded from the refinement.

Results

Effect of Halide Ions on the Absorption Spectrum of TPQ_sq

It is well established that the absorption spectrum of the bound cofactor TPQ changes rapidly during the reductive half-reaction of CAOs (1–3), as demonstrated by stopped-flow spectrophotometry of the reaction with AGAO (29, 40, 53). The final form of TPQ in the reductive half-reaction without turnover (measured under anaerobic conditions) is TPQ_sq, which exhibits characteristic absorption maxima at about 440 and 470 nm with a shoulder at about 350 nm. We incidentally noted that the TPQ_sq spectrum of AGAO was markedly depressed in the presence of NaCl, which was added to the reaction mixture to maintain a constant ionic strength. For example, in the presence of 10 mm NaCl, the intensities of the absorption bands at 352, 438, and 468 nm decreased to about 32% of those without NaCl (Fig. 1). When calculated using the molar extinction coefficient at 468 nm (ϵ₄₆₈ = 4500 m⁻¹ cm⁻¹) reported for TPQ_sq (35), the presence of NaCl reduced the calculated TPQ content relative to total AGAO from 38 to 9.8%. This was attributed to a shift of the equilibrium between TPQ_amr and TPQ_sq (see Scheme 1) toward the former, which has no absorption bands above 300 nm (54). To identify the chemical species that caused this equilibrium shift, we investigated the effects of adding 10 mm NH₄Cl, KCl, Na₂SO₄, or NaH₂PO₄ in the reductive half-reaction and found that comparable shifts were induced by NH₄Cl and KCl but not Na₂SO₄ or NaH₂PO₄ (Fig. 1), clearly indicating that the Cl⁻ ion was preventing the formation of TPQ_sq. Other halide ions (F⁻, Br⁻, and I⁻) behaved similarly (Fig. 2), and their effects were roughly concentration-dependent, with an order of effectiveness of Cl⁻ ≈ Br⁻ > F⁻ > I⁻. A similar effect (bleaching of TPQ_sq absorption) by a high concentration of Cl⁻ (0.45 m KCl) was reported in an early study on methylamine oxidase from Arthrobacter P1 (55), but the mechanism of bleaching was not further pursued.

FIGURE 1.

Effects of salts on TPQ_sq formation. UV-visual absorption spectra of TPQ_sq generated by anaerobic reaction with 2-PEA (1 mm) as the substrate were measured with 100 μm AGAO monomer in 100 mm HEPES, pH 6.8, in the absence (black solid line) and presence of various salts at a final concentration of 10 mm: NaCl (green solid line), NH₄Cl (blue solid line), KCl (blue broken line), Na₂SO₄ (red broken line), or NaH₂PO₄ (red solid line).

FIGURE 2.

Effects of halide ion concentration on TPQ_sq formation. UV-visual absorption spectra of TPQ_sq (generated from 100 μm AGAO monomer and 1 mm 2-PEA) were measured in 100 mm HEPES, pH 6.8, containing 0 (black), 5 (blue), 10 (green), 30 (orange), and 50 mm (red) NaF (A), NaCl (B), NaBr (C), or NaI (D).

Effect of Halide Ions on Catalytic Activity

We have briefly reported the inhibition of AGAO activity by Cl⁻ ion (56) in the past. In this work, more precise steady-state kinetic analyses were conducted by systematically varying the concentrations of the substrate amine (2-PEA) or dissolved oxygen in the presence of 0–50 mm solutions of different halide ions. Double reciprocal plots (1/v versus 1/s) revealed that the Cl⁻ ion was an uncompetitive inhibitor with respect to the substrate amine and a noncompetitive inhibitor with respect to dissolved oxygen (Fig. 3); other halide ions (F⁻, Br⁻, and I⁻) exhibited similar inhibition patterns (data not shown). These results show that halide ions bind to a substrate·enzyme complex but not to the free enzyme and that they bind equally to the O₂-bound and O₂-unbound enzyme forms at a site distinct from the O₂-binding site (41). The K_i values calculated for uncompetitive inhibition were 97.2 ± 6.5 mm for F⁻, 26.2 ± 1.4 mm for Cl⁻, 58.2 ± 2.8 mm for Br⁻, and 69.6 ± 9.4 mm for I⁻, whereas that for noncompetitive inhibition by Cl⁻ was 32.8 ± 1.7 mm. The inhibition of catalytic activity by halide ions may be unique to AGAO; the Cl⁻ ion reportedly has no inhibitory effect on the activities of human kidney diamine oxidase, P. pastoris amine oxidase, or P. savitum CAO (57).

FIGURE 3.

Steady-state kinetics analysis in the presence of NaCl. A, AGAO activity was measured at 30 °C in 50 mm HEPES (pH 6.8) in the presence of 0 (○), 10 (□), 30 (▵), or 50 (♢) mm NaCl with systematically varied 2-PEA concentrations. B, AGAO activity was measured in solutions containing 40 μm 2-PEA and 0 (○), 12.5 (□), 25 (▵), or 50 (♢) mm NaCl with systematically varied solvent O₂ concentrations. Each point represents the mean ± S.E. (error bars) from two independent experiments.

Effects of Halide Ions on Transient Kinetics

To identify the substrate·enzyme complex to which the halides bind, we performed transient kinetic analyses of the reductive half-reaction in the presence of 100 mm NaCl, 300 mm NaBr, or 300 mm Na₂SO₄ (as a control for the ionic strength). As reported previously (29, 40, 53), rapid spectral changes associated with changes in the redox and chemical state of TPQ (TPQ_ox → TPQ_ssb → TPQ_psb → TPQ_amr → TPQ_sq) were observed (Fig. 4). We noted that in the presence of 100 mm NaCl or 300 mm NaBr, the TPQ_sq absorption band appeared transiently within ∼117 ms and then gradually declined in intensity to about 30–40% of the value achieved in the absence of halide ions or in the presence of 300 mm Na₂SO₄ (Fig. 4, A and B). The final spectra (at 1023 ms) acquired in the presence of NaCl or NaBr (Fig. 4, C and D) mostly lacked the TPQ_sq-characteristic peaks, suggesting a shift of the equilibrium toward TPQ_amr, as discussed above (Fig. 2, B and C).

FIGURE 4.

Spectral changes during the reductive half-reaction with 2-PEA. UV-visible absorption spectra were recorded after mixing AGAO (100 μm monomer) with 2 mm 2-PEA (A), 2 mm 2-PEA plus 300 mm Na₂SO₄ (B), 2 mm 2-PEA plus 100 mm NaCl (C), or 2 mm 2-PEA plus 300 mm NaBr (D) in 50 mm HEPES buffer, pH 6.8, at 4 °C under anaerobic conditions. The spectra obtained at 0 (red), 2.30, 3.84, 6.4, 8.96, 14.1, 24.3, 44.8, 117, 209, 332, 600, and 1023 ms are shown using darker colors to represent later times. The arrows indicate the direction of the spectral changes. Deduced absorption spectra are shown for TPQ_ox (red), TPQ_ssb (yellow), TPQ_psb (green), TPQ_amr (black), and TPQ_sq (blue) in E, F, G, and H, calculated from the measurements of A (no salt), B (300 mm Na₂SO₄), C (100 mm NaCl), and D (300 mm NaBr), respectively. The absorption spectra for TPQ_amr·X⁻ (cyan) generated during the experiments using 100 mm NaCl (C), and 300 mm NaBr (D) are shown in G and H, although they are defined to be identical to that of TPQ_amr (black). The inset of A shows the time course of the absorbance at 468 nm in the spectral changes of A (no salt, red), B (300 mm Na₂SO₄, black), C (100 mm NaCl, blue), and D (300 mm NaBr, green).

Initially, the multiwavelength data of all spectral changes were fitted to the four-step mechanism connecting TPQ_ox, TPQ_ssb, TPQ_psb, TPQ_amr, and TPQ_sq (Scheme 1) by global analysis as reported previously (29). The spectral changes in the presence of 300 mm Na₂SO₄ (Fig. 4B) were solved to provide rate constants that were essentially identical to those obtained without the salt (Fig. 4A) (Table 2), and the deduced UV-visible absorption spectra (Fig. 4, E and F) of TPQ_ox, TPQ_ssb, TPQ_psb, TPQ_amr, and TPQ_sq were similar to those reported previously (29). However, the spectral changes observed in the presence of 100 mm NaCl or 300 mm NaBr could not be fitted to the four-step model. On the basis of the spectral changes and inhibition mechanism described above, it appeared that halide ions (X⁻) bound to the TPQ_amr state. We therefore proposed a branched model (with the branch connecting TPQ_ox to TPQ_amr·X⁻; Scheme 1), in which the TPQ_amr·X⁻ complex accumulates. The new model provided a reasonable solution to the data fitting of the spectral changes in the presence of NaCl or NaBr. As shown in Table 2, the rate constants of the steps between TPQ_ox and TPQ_sq (k_±1, k_±2, k_±3, and k_±4) were comparable with those observed without halide ions, although the k₋₃ value was approximately halved. The rate constant of the branching step from TPQ_amr to TPQ_amr·X⁻ (k₊₅) (Scheme 1) was estimated to be half that for TPQ_sq formation (k₊₄). The magnitude of these parameters well explains the slow accumulation of TPQ_amr·X⁻; TPQ_sq is formed initially but gradually converted to TPQ_amr·X⁻ via TPQ_amr. The deduced absorption spectra of TPQ_ox, TPQ_ssb, TPQ_psb, TPQ_amr, and TPQ_sq in the presence of NaCl (Fig. 4G) and NaBr (Fig. 4H) were essentially identical to those observed without halide ions (Fig. 4E) and in the presence of 300 mm Na₂SO₄ (Fig. 4F). Altogether, these results show that halide ions bind to the TPQ_amr form in the reductive half-reaction, thereby inhibiting the formation of TPQ_sq from TPQ_amr.

TABLE 2.

Kinetic constants for each step of the reductive half-reaction in the absence and presence of various salts and a viscogen at 4 °C

	No addition	+300 mm Na2SO4	+100 mm NaCl	+300 mm NaBr	+30% (w/v) glycerol
k+1 (s−1)a	887 ± 0.2	880 ± 0.3	887 ± 0.6	880 ± 0.08	871 ± 0.03
k−1 (s−1)	531 ± 0.8	529 ± 0.05	530 ± 0.2	528 ± 2	560 ± 0.02
k+2 (s−1)	206 ± 0.04	207 ± 0.1	206 ± 0.1	207 ± 0.3	201 ± 0.006
k−2 (s−1)	34 ± 0.008	34 ± 0.02	34 ± 0.02	34 ± 0.07	34 ± 0.001
k+3 (s−1)	102 ± 0.06	102 ± 0.05	102 ± 0.06	103 ± 0.2	100 ± 0.003
k−3 (s−1)	127 ± 8	127 ± 5	63 ± 3	76 ± 7	134 ± 0.007
k+4 (s−1)	39 ± 9	39 ± 6	32 ± 6	23 ± 11	15 ± 0.001
k−4 (s−1)	17 ± 3	12 ± 2	15 ± 4	21 ± 6	6 ± 0.001
k+5 (s−1)a			15 ± 7	21 ± 12
k−5 (s−1)			5 ± 2	9 ± 3

^a Determined as a first-order rate constant at constant and excess concentrations of amine substrate (2-PEA) and halide ions.

Effect of Solvent Viscosity on AGAO Activity

To further probe the equilibrium shift between TPQ_amr and TPQ_sq, we examined the dependence of catalytic activity on solvent viscosity, which can perturb diffusion-controlled steps, including substrate binding, product release, and conformational changes of the enzyme (58, 59). Glycerol (M_r = 92.1) and sucrose (M_r = 342.3) were used as viscogens. Steady-state kinetic analyses in viscogenic solutions, in which the relative solvent viscosity (η/η⁰, where η and η⁰ denote viscosities in the presence and absence of the viscogen, respectively) was increased by adding glycerol or sucrose, yielded values of k_cat/K_m for 2-PEA that were basically identical to that observed in non-viscogenic solution, whereas the k_cat values decreased. The ratio of the viscogen-free k_cat value to that in the presence of viscogen (k_cat⁰/k_cat, where k_cat⁰ and k_cat denote the rate constants in the absence and presence of the viscogen, respectively) was roughly proportional to the relative solvent viscosity (Fig. 5). This suggests that a diffusion-controlled step(s) is included in the overall reaction consisting of TPQ_imq, TPQ_ssb, TPQ_psb, TPQ_amr, TPQ_sq, and TPQ_imq under steady-state conditions (Scheme 1).

FIGURE 5.

Dependence of steady-state kinetic parameters on solvent viscosity. The ratios of k_cat/K_m (A) and k_cat (B) in the presence and absence of viscogen at 4 °C were plotted against the relative solvent viscosity, η/η⁰, where zero denotes the value in the absence of viscogen. Black circles and squares, data points for glycerol and sucrose, respectively. A thin black line denotes the theoretical result for a completely diffusion-controlled process with a slope of 1.0. Error bars, S.D. (n = 2).

To evaluate the effect of solvent viscosity on each step of the reductive half-reaction, transient kinetic analyses were conducted (Fig. 6). Stopped-flow measurements in the absence and presence of 30% (w/v) glycerol generally produced similar spectral changes, but the formation of TPQ_sq was clearly slower in the viscogen's presence (Fig. 6, A and B, insets). Slow TPQ_sq formation was evident from the comparison of the traces of absorbance changes at 468 nm specific to TPQ_sq (Fig. 6C). Further, the rate constants of each step (k₊₁, k₊₂, k₊₃, k₋₁, k₋₂, and k₋₃) in the reductive half-reaction were determined by global analysis according to the four-step model starting from TPQ_ox and ending at TPQ_sq (Scheme 1). Of these rate constants, k₊₁, k₊₂, k₊₃, k₋₁, k₋₂, and k₋₃ were independent of the glycerol concentration, but the values of k₊₄ and k₋₄ (which relate to the interconversion of TPQ_amr and TPQ_sq) decreased in proportion to the glycerol concentration (Table 2), showing that this step is diffusion-controlled. For detailed analysis, the ratios of k⁰/k were plotted against the relative solvent viscosity, η/η⁰ (Fig. 6D), where k⁰ and k are the rate constants (k₊₂, k₋₂, k₊₄, and k₋₄) in the absence and presence of viscogen (glycerol), respectively. The ratios of k₊₄⁰/k₊₄ and k₋₄⁰/k₋₄ increased significantly in proportion to the relative solvent viscosity, although the slope of the fitted line (0.50) indicated a partial effect; an entirely diffusion-controlled reaction would give a slope of 1.0 (60) (Fig. 6D). The ratios of the other rate constants were almost independent of the relative solvent viscosity (e.g. slope = 0.003 for k₊₂⁰/k₊₂) (Fig. 6D). The reaction step connecting TPQ_amr and TPQ_sq involves neither substrate binding nor product release. Thus, these findings show that the interconversion of TPQ_amr and TPQ_sq is accompanied by a conformational change(s) of the enzyme. Reducing the viscogen's access to the region undergoing the conformational change could reduce its effective concentration, explaining the partial effect indicated by the k⁰/k slope of ∼0.5. It is therefore likely that the conformational change(s) occurs somewhere in the protein interior, such as the buried active site (most likely in the TPQ cofactor itself). Supporting this, we were unable to observe any effect of solvent viscosity on the rate constants k₊₄ and k₋₄ when using 30% (w/v) sucrose as the viscogen; sucrose's molecular size is 3.7-fold greater than that of glycerol, so it probably cannot penetrate into the active site cavity (Fig. 6D).

FIGURE 6.

Effects of viscosity on the reductive half-reaction. UV-visible absorption spectra were recorded in 50 mm HEPES buffer, pH 6.8, at 4 °C under anaerobic conditions upon mixing AGAO (100 μm monomer) with 2 mm 2-PEA (A) or 2 mm 2-PEA (B) in the presence of 30% (w/v) glycerol. The spectra obtained at 0 (broken line), 2.30, 3.84, 6.4, 8.96, 14.1, 24.3, 44.8, 117, 209, 332, 600, and 1023 ms are shown with darker colors representing later times. The arrows indicate the direction of the spectral changes. The insets expand the 440–500 nm region for easier inspection of the spectral changes. C, absorbance at 468 nm was monitored during the spectral change of 2 mm 2-PEA (broken line) (A) or 2 mm 2-PEA (B) in the presence of 30% (w/v) glycerol (solid line) at 0–0.6 s. D, ratios of the rate constants (k₊₂⁰/k₊₂ (red open circle), k₋₂⁰/k₋₂ (red open triangle), k₊₄⁰/k₊₄ (red triangle), and k₋₄⁰/k₋₄ (red rhombus) determined in solutions of up to 30% (w/v) glycerol and k₊₂⁰/k₊₂ (black open circle), k₋₂⁰/k₋₂ (black open square), k₊₄⁰/k₊₄ (black open triangle), and k₋₄⁰/k₋₄ (black open rhombus) determined in solutions of up to 30% (w/v) sucrose) were plotted against the relative viscosity. S.D. values were less than 0.03% at all points (n = 2–4) and therefore are not shown with error bars. Thick lines (black and red) denote fitted linear lines for the k₊₄⁰/k₊₄ values for sucrose and glycerol, respectively. The gray solid line denotes the theoretical result for a completely diffusion-controlled process with a slope of 1.0.

Reductive Half-reaction in Crystals

To obtain structural insights into the interconversion between TPQ_amr and TPQ_sq, the AGAO crystals were reacted anaerobically with substrates in the presence or absence of halide ions under six different conditions (Table 3), and the intermediates formed were freeze-trapped for structural determination. Before x-ray analysis, these crystals were subjected to single-crystal microspectrophotometry to identify the reaction intermediates present within them (29, 40, 41). The absorption spectra of the frozen crystals anaerobically soaked with 4 mm 2-PEA or 10 mm histamine (these crystals were designated AGAO_PEA and AGAO_HTA, respectively) (Fig. 7, A and B) were very similar to that of TPQ_sq for the enzyme in solution, showing specific absorption peaks around 365, 438, and 465 nm (Fig. 1). In contrast, the crystals anaerobically soaked with 50 mm ethylamine (hydrochloride) (AGAO_ETA/HCl) gave a rather peakless spectrum resembling that of TPQ_amr (Fig. 7C), although small TPQ_sq-like peaks remained. Furthermore, in the presence of 100 mm NaCl or 300 mm NaBr, the crystals prepared by anaerobic soaking with 4 mm 2-PEA (designated AGAO_PEA/NaCl and AGAO_PEA/NaBr, respectively) exhibited absorption spectra (Fig. 8, A and B) that were distinct from those of TPQ_sq and TPQ_amr but comparable with that of TPQ_psb for the enzyme in solution (Fig. 4E). To identify the halide ion-binding site(s) in the unreacted enzyme, the crystal was aerobically soaked with 300 mm NaBr alone (AGAO_NaBr). This yielded a spectrum identical to that of TPQ_ox because the reaction could not proceed (Fig. 8C). These crystal absorption spectra revealed that the reductive half-reaction occurred in all of the crystals except for AGAO_NaBr and that the accumulation of the TPQ_psb, TPQ_amr, or TPQ_sq intermediates could be induced by soaking the crystals with appropriate amine substrates in the presence or absence of halides (Table 3).

TABLE 3.

Summary of crystal soaking conditions and active-site features determined by x-ray crystallographic analysis

	AGAOPEA	AGAOHTA	AGAOETA/HCla	AGAOPEA/NaCl	AGAOPEA/NaBr	AGAONaBr
Soaking conditions	2-PEA, anaerobic	Histamine, anaerobic	Ethylamine, anaerobic	2-PEA, NaCl, anaerobic	2-PEA, NaBr, anaerobic	NaBr, aerobic
Assigned TPQ species	TPQsq	TPQsq	TPQamr	TPQpsb	TPQpsb	TPQox
Conformation of TPQ	On copper	On copper	Off copper	Off copper	Off copper	Off copper
Copper coordination geometry	Tetrahedral	Tetrahedral	Square pyramidal	Square pyramidal	Square pyramidal	Square pyramidal
Presumed copper valence	Cu(I)	Cu(I)	Cu(II)	Cu(II)	Cu(II)	Cu(II)
Axial ligand of copper atom (distance, Å)	TPQsq C4-OH (2.8)	TPQsq C4-OH (2.7)	Cl− (2.5)	Cl− (2.3)	Br− (2.5)	Water (2.6)

^a Data for chain A.

FIGURE 7.

Catalytic intermediate structures and UV-visible absorption spectra of AGAO after reaction with 2-PEA, histamine, and ethylamine hydrochloride. AGAO crystals were anaerobically soaked with excess 2-PEA, histamine, or ethylamine hydrochloride, and the x-ray structures of the resulting crystals were determined. UV-visible absorption spectra for single crystals of AGAO_PEA (A), AGAO_HTA (B), and AGAO_ETA/HCl (C) were measured before x-ray exposure. The active-site structures of AGAO_PEA (D), AGAO_HTA (E), and AGAO_ETA/HCl (F) are shown superimposed on the F_o − F_c omit map (blue mesh) for residue 382 contoured at 3.5 σ. Active-site residues are represented by green stick models. Water molecules and copper centers are represented by brown and cyan spheres, respectively. All molecular drawings were generated using PyMOL.

FIGURE 8.

Active-site structures and UV-visible absorption spectra of AGAO soaked with halide salts and/or substrates. AGAO crystals were anaerobically soaked with excess 2-PEA in the presence of NaBr (A and D) or NaCl (B and E). C and F show results for AGAO crystals soaked only with NaBr under aerobic conditions. UV-visible absorption spectra for the single crystals of AGAO_PEA/NaBr (A), AGAO_PEA/NaCl (B), and AGAO_NaBr (C) were measured before x-ray exposure. The refined model of the active site structures of AGAO_PEA/NaBr (D), AGAO_PEA/NaCl (E), and AGAO_NaBr (F) are shown superimposed on the F_o − F_c omit map (blue mesh) for residue 382 contoured at 3.5 σ. Bromine-anomalous maps contoured at 8 σ are represented by red meshes in the active sites of AGAO_PEA/NaBr (D) and AGAO_NaBr (F). The anomalous dispersion of the copper atom is pronounced although the wavelength of the used x-ray (0.919 Å) deviated from the peak wavelength of the copper atom (1.3808 Å), and an anomalous peak (about 18 σ) was detected on the copper site of the active center as well as for the bromine atom. Active-site residues are represented by green stick models. Water molecules and copper atoms are represented by brown and cyan spheres, respectively. All molecular drawings were generated using PyMOL.

The overall structures determined for AGAO_PEA, AGAO_HTA, AGAO_ETA/HCl, AGAO_PEA/NaCl, AGAO_PEA/NaBr, and AGAO_NaBr were comparable with that of resting AGAO (Protein Data Bank code 1IU7), with root mean square deviations for the main-chain atoms within ∼0.4–0.5 Å. In these crystal structures, we assigned the chemical structures of TPQ based on its absorption spectra (Table 3) and constructed the active-site structures using the assigned models of TPQ in different conformations, which were built to coincide with the F_o − F_c omit maps (Figs. 7 and 8). The two monomers (chains A and B) of the homodimer in the asymmetric unit of the crystals showed essentially identical active-site structures except for those in the AGAO_ETA/HCl crystal, in which active-site residues, including TPQ and water molecules, had slightly different conformations and electron densities between the two monomers.

The most notable finding from the crystal structures was that the TPQ_sq moieties in AGAO_PEA and AGAO_HTA had an on-copper conformation with ∼100% occupancy, with the 4-OH group of TPQ_sq projecting toward the copper center at a distance of 2.7–2.9 Å (Fig. 7, D and E) and the 5-NH₂ group positioned opposite to the catalytic base (Asp-298) in close proximity to Met-602. This is the first x-ray structure of CAO with TPQ_sq being exclusively copper-ligating, although an on-copper TPQ_sq structure with ∼65–70% occupancy was recently reported for HPAO-1 crystals reduced with methylamine in a low oxygen environment at pH 8.5 (39). The copper center in AGAO_PEA and AGAO_HTA was tetrahedrally coordinated with the 4-OH group of TPQ at the “axial” position and the imidazole groups of three histidines (His-431, His-433, and His-592) at the “equatorial” positions. No water molecules were coordinated to the copper center. On the other hand, TPQ_amr in AGAO_ETA/HCl had an off-copper conformation with ∼100% occupancy, in which the 5-NH₂ group of TPQ_amr was positioned close to Asp-298 and the 4-OH group was hydrogen-bonded to the Tyr-284 side chain rather than coordinating to the copper center (Fig. 7F). The off-copper conformation was the sole conformer in chain A. However, both the off-copper and on-copper conformers were present (at a relative abundance of about 6:4) in chain B. Probably, the minor on-copper conformation in chain B was partly responsible for the crystals' small TPQ_sq-like absorption peaks (Fig. 7C). The spectrophotometrically assigned TPQ_psb moiety of AGAO_PEA/NaCl and AGAO_PEA/NaBr also had an off-copper conformation, in which the C5 position of TPQ was connected to additional electron density corresponding to the phenylethyl moiety of the product phenylacetaldehyde (PAA) via a covalent linkage in the form of an imine bond (Fig. 8, D and E).

The binding of several halide ions was detected in the AGAO_ETA/HCl, AGAO_PEA/NaCl, AGAO_PEA/NaBr, and AGAO_NaBr crystals (2 and 5 Cl⁻ ions/dimer in AGAO_ETA/HCl and AGAO_PEA/NaCl, respectively, and 8 Br⁻ ions/dimer in AGAO_PEA/NaBr and AGAO_NaBr), as judged by the anomalous peaks (over 7 σ) generated by the Br⁻ ion and the electron densities (over 8 σ) of the Cl⁻ ion, which were clearly greater than those of water molecules (less than 5 σ). The Cl⁻ ions identified in AGAO_ETA/HCl derived from the substrate (ethylamine hydrochloride), which was present at a high concentration (50 mm). In the halide-bound complexes, a Br⁻ or Cl⁻ ion was found to occupy the axial coordination site of the active-site copper center in the AGAO_ETA/HCl, AGAO_PEA/NaCl, and AGAO_PEA/NaBr structures (Figs. 7F and 8 (D and E) and Table 3). These are the first x-ray crystal structures of CAO with an anionic inhibitor (57, 61, 62) bound to the active site copper center. In contrast, the axial position in AGAO_NaBr was occupied by a water molecule rather than Br⁻ (Fig. 8F and Table 3); Br⁻ ions instead bound to the protein surface in a seemingly nonspecific fashion. Similar behavior was observed in AGAO_PEA/NaBr. Specific binding of a halide ion at the axial position of the active-site copper center only occurred in crystals that had been anaerobically soaked with substrate, strongly suggesting that the uncompetitive inhibition of the steady-state reaction by halide ions with respect to 2-PEA is due to their ability to bind to the copper center in the reaction intermediates, in which TPQ is reduced with substrates, rather than to the copper center in the free enzyme. It also suggests that the axial ligand-binding position of the copper center exhibits a stronger preference for halide ions in the substrate-reduced form of AGAO than in the resting form.

Comparison of the on-copper TPQ_sq structures of AGAO_PEA and AGAO_HTA with the off-copper TPQ_ox structure in resting AGAO (Protein Data Bank code 1IU7) revealed that most active-site residues (except for Tyr-296 and Met-602), water molecules, and the copper center are retained in almost the same positions and conformations (Fig. 9). In the TPQ_sq structures, the side-chain phenol ring of Tyr-296 rotates ∼80° around the Cα–Cβ bond to participate in a hydrogen-bonding network involving two water molecules (W1 and W2); the 2-OH, 4-OH, and 5-NH₂ groups of TPQ_sq; the 4-OH group of Tyr-284; and the Sδ atom of Met-602 (Fig. 9A). The on-copper conformation is probably stabilized by this hydrogen-bonding network, allowing the direct coordination of the 4-OH group of TPQ to the copper center, which would facilitate rapid, ligand-to-metal charge transfer-like electron transfer from TPQ_amr to Cu(II) to form the TPQ_sq·Cu(I) state.

FIGURE 9.

Interactions in the on-copper and off-copper conformations. The active sites of AGAO_PEA (A) and the substrate-free and oxidative form of AGAO (Protein Data Bank code 1IU7) (B), in which the TPQ ring has on-copper and off-copper conformations, respectively, are drawn showing hydrogen bonds (dotted lines) and ligation to the copper centers (red lines). Estimated hydrogen bond lengths are shown in Å. The superposition of A and B is shown in C, in which the on-copper (A) and off-copper (B) structures are colored in purple and gray, respectively. All molecular drawings were generated using PyMOL.

In keeping with the single-crystal microspectrophotometric observations, a TPQ_psb-like intermediate was identified in the AGAO_PEA/NaBr and AGAO_PEA/NaCl structures, which was probably formed by a condensation reaction between the amino group of TPQ_amr (in the off-copper form) and the aldehyde group of the reaction product PAA, which remains in the substrate-binding hydrophobic pocket (Fig. 8, D and E); PAA was indeed found to remain bound in the AGAO_PEA structure (Fig. 7D), as reported previously for the 2-PEA-reduced E. coli CAO crystals (63). This assumption is also supported by the finding that the TPQ_psb observed in AGAO_PEA/NaBr and AGAO_PEA/NaCl had a cis-configuration, whereas the TPQ_psb formed during the reductive half-reaction of the D298A mutant of AGAO had a trans-configuration (29). The absence of the product aldehydes (imidazole-4-acetaldehyde and acetaldehyde) in the AGAO_HTA and AGAO_ETA/HCl structures is probably due to their low affinities for the substrate-binding pocket of AGAO; for comparative purposes, the K_m values for histamine (1.2 mm) and ethylamine (170 mm) are 220- and 30,000-fold higher, respectively, than that for 2-PEA (5.4 μm) (41). Overall, these findings indicate that in the AGAO_PEA/NaBr and AGAO_PEA/NaCl crystals, the binding of halide ions (Br⁻ and Cl⁻) to the axial position of the copper center prevented TPQ_amr from adopting the on-copper conformation, causing it to back-react with PAA to form a TPQ_psb configuration distinct from that formed during the reductive half-reaction.

Effect of pH on the Equilibrium between TPQ_amr and TPQ_sq

The equilibrium between TPQ_amr and TPQ_sq in AGAO has been reported to shift toward TPQ_sq under alkaline conditions (35), which suggests that an ionizable group(s) plays a role in triggering the conformational change of the cofactor. We therefore used spectrophotometry to investigate the effect of pH on the equilibrium between TPQ_amr and TPQ_sq. The reductive half-reaction was performed with 2-PEA as the substrate at pH values ranging from 5.7 to 10.1 with a constant ionic strength (Fig. 10A); AGAO is stable in this pH range. The TPQ_sq-specific absorption peaks at about 440 and 470 nm, and the shoulder at about 350 nm increased in intensity as the pH rose from 5.7 to 8.5 but did not change further above pH 8.5, indicating that the equilibrium shifted toward TPQ_sq above pH 8.5. By plotting the absorbance at 468 nm, to which TPQ_amr makes no contribution, against the pH (Fig. 10B) and fitting the data to Equation 1, two ionizable groups with apparent pK_a values of 5.96 ± 0.05 (pK_a1) and 7.74 ± 0.19 (pK_a2) were found to be involved in the equilibrium shift from TPQ_amr to TPQ_sq. Judging from the magnitude of pH-independent absorbance values (C₁ = 0.164, C₂ = 0.053), deprotonation of the ionizable group with pK_a = 5.96 contributes predominantly to the equilibrium shift.

FIGURE 10.

pH dependence of TPQ_sq formation. A, 100 μm AGAO monomer was anaerobically reduced with 1 mm 2-PEA at various pH values in the presence of 100 mm Na₂SO₄, and UV-visual absorption spectra of AGAO were measured at 25 °C after a 5-min preincubation. B, absorbance at 468 nm specific to TPQ_sq was plotted against pH. A solid line indicates the theoretical line obtained by data fitting. The spectra at pH 5.67, 6.02, 6.53, 6.99, 7.52, 8.03, 8.54, 9.04, 9.55, and 10.12 are shown with darker colors corresponding to higher pH values. Each point represents the mean ± S.E. (error bars) from 2–4 independent experiments.

Discussion

The results presented above demonstrate that the off-copper and on-copper conformations of TPQ are readily interconvertible during the reductive half-reaction with various amine substrates and in the presence of halide ions. In the steps prior to the formation of TPQ_amr (Scheme 1), the catalytic reaction proceeds with TPQ always maintained in the off-copper conformation irrespective of its chemical state (29, 40, 41). In the TPQ_ssb and TPQ_psb states, the distal part of the substrate amine is anchored to the substrate-binding pocket, preventing the TPQ ring from adopting the on-copper conformation (29). However, once TPQ_psb is hydrolyzed, the reduced TPQ gains the conformational flexibility that enables facile interconversion between the off-copper and on-copper conformations.

In the on-copper conformation of TPQ_sq as observed in the AGAO_PEA and AGAO_HTA structures (Fig. 9, A and B), the active-site copper center is equatorially coordinated by three imidazole groups from His residues without the equatorial water ligand seen in the resting TPQ_ox·Cu(II) state (17) and other intermediates formed in the reductive half-reaction (Figs. 7 and 8). A similar decrease in the number of equatorial ligands at the copper center was observed in extended x-ray absorption fine structure studies on various dithionite-treated CAOs, in which Cu(II) is reduced to Cu(I) (64). It is therefore suggested that the tetrahedrally coordinated copper centers observed in the AGAO_PEA and AGAO_HTA crystals are probably in the Cu(I) oxidation state, as was proposed in a recent paper on the structure of methylamine-reduced HPAO-1 (39). Based on the full occupancy of the modeled TPQ_sq in the electron density map, essentially all of their TPQ is assumed to be in the TPQ_sq state. Taken together, these results show that the on-copper conformation is a consequence of 1e⁻ transfer from TPQ_amr to Cu(II) to form the TPQ_sq·Cu(I) state. In contrast, a water molecule (W_eq) was identified as an equatorial ligand of the copper atom in the AGAO_ETA/HCl crystal, with observed electron densities of 3.7 and 2.3 σ in crystallographically distinguishable chains A and B, respectively. In addition, a Cl⁻ ion was found to occupy an axial position in the copper complex (Fig. 7C), whose five-coordinate square pyramidal structure suggests that the copper is in the Cu(II) oxidation state, especially in chain A, for which TPQ is mostly in the off-copper TPQ_amr state based on its absorption spectrum and x-ray structure. We have so far observed neither the off-copper TPQ_sq·Cu(I) form nor the on-copper TPQ_amr·Cu(II) form, strongly suggesting that the 1e⁻ transfer occurs exclusively in the on-copper conformation of TPQ_amr. This also leads to the suggestion that before the formation of the TPQ_sq·Cu(I) state, the TPQ quinone ring moves from the off-copper to the on-copper conformation.

The spectroscopic data from the transient kinetics experiments showed that the formation of TPQ_sq is dependent on solvent viscosity (Fig. 6D), further supporting the occurrence of a conformational change in TPQ during the last step of the reductive half-reaction. Because the rate constants of this step (k_±4) were determined from the absorbance changes associated with TPQ_sq, they represent the rates of both the conformational change of TPQ and the subsequent electron transfer from TPQ_amr to Cu(II). The same presumption holds for the rate constants of the electron transfer (k_ET) determined previously by temperature-jump relaxation studies (33, 35, 65), which varied from 60–75 s⁻¹ (Arthrobacter P1 methylamine oxidase) (65) to 20,000 s⁻¹ (P. savitum CAO) (33). The latter extremely large rate constant was attributed to the intrinsic k_ET and interpreted to mean that the TPQ cofactor is in close proximity (∼3 Å) to the copper center (33) (cf. the distance of 2.6 Å between the TPQ 4-O atom and the copper center; Fig. 9). It is thus likely that the smaller rate constants determined previously (k_ET) and in this study (k₊₄) mainly reflect the rate constants for the conformational change of the TPQ cofactor, in agreement with the suggestion that conformationally “gated” or controlled electron transfer is plausible (33). An extended x-ray absorption fine structure study (64) also raised the possibility that variations in the redox potentials or the effective electron transfer distance between TPQ_amr and Cu(II) may control k_ET. The difference between the rate constants for AGAO determined here (Table 2; k₊₄ = 39 s⁻¹ at 4 °C, pH 6.8) and previously (k_ET = 73 s⁻¹ at 5 °C, pH 7.2) (37) is probably due to subtle differences in temperature and pH, both of which strongly affect the equilibrium between TPQ_amr·Cu(II) and TPQ_sq·Cu(I) (33, 35, 37, 39, 65) (Fig. 10); at higher pH values and temperatures, the equilibrium shifts toward TPQ_sq·Cu(I), which suggests a ΔH value of >0 for the conformational change of TPQ. Finally, it should be noted that the rate constants of the conformational change are appreciably larger than the k_cat value (17 s⁻¹) determined by steady-state kinetics under the same conditions (at 4 °C and pH 6.8), supporting the hypothesis that the conformational change of the cofactor can occur within the overall turnover reaction.

Based on the pH dependence of the equilibrium between TPQ_amr and TPQ_sq, it is suggested that deprotonation of two ionizable groups with pK_a values of 5.96 ± 0.05 and 7.74 ± 0.19 (Fig. 10B) facilitates the equilibrium shift toward TPQ_sq. Among several ionizable groups in the active site (5-NH₂ (estimated pK_a, 5.88), 4-OH (9.59), and 2-OH (11.62) groups of TPQ_amr (54); the carboxyl group of Asp-298 (7.5 ± 0.20) (29); and the water axially coordinated to the copper atom (∼7.5) (66)), the 5-NH₂ group of TPQ_amr is most likely assigned to the group with the lower pK_a value (5.96) and mainly contributes to the equilibrium shift toward TPQ_sq, with the neutral form of TPQ_amr that is the major form at pH >7 being the direct precursor to TPQ_sq (Scheme 1). Furthermore, the neutral form of TPQ_amr is only weakly tethered in the active site, forming neither electrostatic interactions nor charge-assisted hydrogen bonds (67) with surrounding residues. It should thus be amenable to facile conformational change. The ionizable group with the higher pK_a value (7.74), although contributing insignificantly to the equilibrium shift toward TPQ_sq, may be ascribed to the 4-OH group of TPQ_sq with a pK_a value of 6.39 determined with a model compound (68), rather than the same group in TPQ_amr (pK_a 9.59) (54); deprotonation of the 4-OH group of TPQ_sq is expected to stabilize the TPQ_sq form (see Scheme 1). If this is the case, deprotonation of the 4-OH group would occur after the conformational change of the TPQ ring and the electron transfer from TPQ_amr to Cu(II). A notable increase of the pK_a value (from 6.39 to 7.74) may be conceivable to occur in the hydrophobic active site of CAOs, as observed for the carboxyl group of the catalytic base Asp-298 with a significantly high pK_a value (7.5 ± 0.20) (29).

The TPQ_amr structures have been determined previously with the substrate-reduced forms of E. coli CAO and HPAO-1 (Protein Data Bank codes 1D6U and 4EV2, respectively), in which the axial positions of the copper atom are occupied by water and a dioxygen species, respectively. Comparison of these structures with the TPQ_amr of AGAO bound with a chloride ion (AGAO_ETA/HCl) has revealed that the TPQ_amr ring is tilted anticlockwise by about 20° (rigid body rotation around the Cβ–Cγ bond) in AGAO (Fig. 11A). This ∼20° tilting of the TPQ ring appears to result from the minuscule movement (by 0.4 Å) of the position of the 2-OH group of TPQ_amr, probably due to the repulsion from the axially coordinated chloride ion that has a larger van der Waals radius than water and a dioxygen species. Consequently, the 5-NH₂ group approaches within hydrogen bond distance (2.8 Å) to the carboxyl group of Asp-298 (Fig. 11A), which is predominantly protonated at crystallization pH of 6.8, thereby lowering the nucleophilicity of the 5-NH₂ group. The hydrogen bond may also stabilize the tilted conformation of TPQ_amr even after the halide ion is released (in the step of k₋₅ in Scheme 1). In addition, the aldehyde group of the product PAA that remains bound in AGAO_PEA is located rather distant (3.2 Å) from the 5-NH₂ group of TPQ_amr in the AGAO_ETA/HCl structure to undergo the nucleophilic attack (Fig. 11B). These structural consequences well explain the significantly decreased rate constant (k₋₃) for the back-formation of TPQ_psb in the presence of halide ions (Table 2). Moreover, the geometry of the 5-NH₂ group of TPQ_amr relative to the aldehyde carbon atom of PAA strongly suggests that the nucleophilic attack occurs from the si face of the carbonyl carbon, leading to the formation of TPQ_psb in cis-configuration (cis-TPQ_psb) (Fig. 11D), unlike the formation of TPQ_psb in trans-configuration (trans-TPQ_psb) from TPQ_ssb in the forward reductive half-reaction of the D298A mutant (Fig. 11C) (29).

FIGURE 11.

Effect of binding of chloride ion at the axial position of the copper center on the conformation of TPQ_amr. A, conformation of the TPQ_amr ring in the AGAO_ETA/HCl structure (green) is compared with those of the substrate-reduced E. coli CAO (ECAO) (purple) and HPAO-1 (magenta). Cyan spheres, copper atoms. Residue numbers are referred to those of AGAO. van der Waals surfaces of the chloride ion and the oxygen atom of the 2-OH group of TPQ_amr are represented with gray dots. B, comparison of cis-TPQ_psb formed in AGAO_PEA/NaCl (orange), cis-TPQ_psb formed in the D298A mutant of AGAO (29) (gray), TPQ_amr formed in AGAO_ETA/HCl (green), and PAA formed in AGAO_PEA (green). C, schematic drawing of the presumed mechanism of the formation of cis-TPQ_psb in AGAO_PEA/NaCl.

Monovalent anions, such as cyanide and azide, were reported to be inhibitors of various CAOs, showing competitive, noncompetitive, uncompetitive, or mixed type inhibition with respect to the amine substrate and dioxygen (57, 61, 62). For AGAO, cyanide is an uncompetitive inhibitor with respect to the amine substrate, and azide is a noncompetitive inhibitor with respect to both amine and dioxygen (57, 61). Although the inhibition patterns of halide ions (uncompetitive and noncompetitive with respect to amine substrate and dioxygen, respectively) (Fig. 3) for AGAO are similar to those of azide and cyanide, their effects on the equilibrium between TPQ_amr and TPQ_sq are very different. Halide ions inhibit TPQ_sq formation (Fig. 2) by axially coordinating to the copper center (Figs. 7F and 8 (D and E)), whereas azide converts TPQ_sq into a ligand-to-metal charge transfer complex (57), and cyanide facilitates TPQ_sq formation (61), both of which were suggested to bind at an equatorial position of the copper center (69, 70). It is assumed that azide is probably too large to bind axially to the copper atom and probably cyanide too. Although we cannot currently explain why halide ions, but not azide/cyanide, bind at the axial position of the copper center in the reduced form of TPQ and inhibit TPQ_sq formation, it is reasonable to assume that halide ion binding at the axial position of the copper atom would block the coordination of the 4-OH group of TPQ_amr to the copper center and prevent the subsequent electron transfer to Cu(II). The inability of halide ions to bind to the axial position of the copper center in the resting TPQ_ox state (Fig. 8F) may be due to electrostatic repulsion from the delocalized negative charge through the 4-O⁻ to 2-C=O group of TPQ_ox; in the TPQ_amr state, both the 2-OH and 4-OH groups (whose estimated pK_a values are 11.62 and 9.59, respectively) (54) are neutral and therefore would not electrostatically repel halide ions. The uncompetitive inhibition of halide ions with respect to the amine substrate is consistent with their binding only to the reaction intermediate, whereas noncompetitive inhibition with respect to dioxygen indicates halide binding at a site distinct from the O₂-binding site. The exclusive binding of halide ions to Cu(II) with TPQ_amr in the off-copper conformation (which does not bind dioxygen) is challenging to reconcile with their activity as inhibitors of the oxidative half-reaction that are noncompetitive with respect to dioxygen. This issue can be resolved by supposing that dioxygen binds directly to Cu(I) with TPQ_sq in the on-copper conformation during the oxidative half-reaction and undergoes 1 e⁻-reduction by Cu(I) through the inner sphere mechanism proposed for AGAO (37, 38). Alternatively, halide ions may inhibit the oxidative half-reaction by binding to the O₂-bound enzyme.

The off-copper to on-copper conformational change of TPQ_amr involves three motions of the TPQ ring: sliding (∼53° rotation around the Cα–Cβ bond), tilting up (∼20° rigid body rotation centered at the Cα carbon), and revolution (180° rotation around the Cβ–Cγ bond) (Fig. 12). In the off-copper conformation, the TPQ ring is sandwiched between the side chains of Asn-381 and Tyr-384/Val-282 in a narrow wedge-shaped space (29, 53). The initial step of the conformational change is a simultaneous combination of sliding and tilting up in order to avoid a steric clash between the TPQ ring and the side chain of His-433, which would come within ∼1 Å of the ring if it only slid. This combined sliding/tilting-up motion leads to the axial coordination of the 4-OH group of TPQ_amr to the copper atom, where there is sufficient space for the TPQ ring to rotate by 180° around the Cβ–Cγ bond. The final 180° rotation of the TPQ ring can only occur in the clockwise direction because anticlockwise rotation would lead to a clash between the 5-NH₂ group and the His-433 side chain while permitting a minor movement of the Tyr-384 side chain (Fig. 12). It is unclear whether the electron transfer from TPQ_amr to Cu(II) occurs immediately upon formation of the on-copper conformation of the TPQ ring or after the 180° rotation to the final conformation stabilized by the hydrogen-bonding network (Fig. 9A).

FIGURE 12.

Possible route for the conformational change of TPQ in the active site of AGAO. Stick models of the active-site residues in AGAO_ETA/HCl (TPQ_amr, cyan) and AGAO_PEA (TPQ_sq, yellow) are shown within the cavity with its surface drawn in half-transparent gray. The on-copper TPQ_amr conformer predicted after the first combined sliding/tilting up motion is colored green. The following 180° rotation of the TPQ ring provides a conformation identical with that of the on-copper TPQ_sq. The rotation direction and movement of the TPQ ring are shown with blue arrows. The figure was generated with PyMOL.

Finally, it is noteworthy that the on-copper TPQ_sq structure stabilizes the conformation of the side chain of Met-602 by hydrogen bond formation between the 5-NH₂ group of TPQ_sq and the Sδ atom of Met-602 (Fig. 9). Met-602 is located at the end of the predicted O₂ pathway from the O₂-prebinding site to the copper center and has conformational flexibility with dual extreme conformers (19). Thus, it is tempting to speculate that the tethering of the Met-602 side chain could act as a gate to allow O₂ to enter into the copper center in the initial phase of the oxidative half-reaction.

In conclusion, the results presented herein show that TPQ undergoes a large conformational change during the reductive half-reaction of AGAO, which efficiently mediates between the acid/base chemistry conducted in the off-copper conformation of TPQ by the conserved catalytic base (Asp-298 in AGAO) and the redox chemistry conducted in the on-copper conformation of TPQ at the metal center.

Author Contributions

T. M., A. H., S. N., T. N., H. Y., K. T., and T. O. participated in research design. T. M., A. H., S. N., M. K., Y. K., and T. O. conducted experiments. All authors performed data analysis and wrote or contributed to the writing of the manuscript.