Crystal Structures and Coordination Behavior of Aqua- and Cyano-Co

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Crystal Structures and Coordination Behavior of Aqua- and CyanoCo(III) Tetradehydrocorrins in the Heme Pocket of Myoglobin Yoshitsugu Morita,† Koji Oohora,†,‡ Eiichi Mizohata,† Akiyoshi Sawada,§ Takashi Kamachi,§ Kazunari Yoshizawa,§,∥ Tsuyoshi Inoue,† and Takashi Hayashi*,† †

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan § Institute for Materials Chemistry and Engineering and International Research Center for Molecular Systems, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan ∥ Elements Strategy Initiative for Catalysts and Batteries (ESICB), Kyoto University, Nishikyo-ku, Kyoto 615-8520, Japan ‡

S Supporting Information *

ABSTRACT: Myoglobins reconstituted with aqua- and cyano-Co(III) tetradehydrocorrins, rMb(Co III(OH2 )(TDHC)) and rMb(CoIII(CN)(TDHC)), respectively, were prepared and investigated as models of a cobalamin-dependent enzyme. The former protein was obtained by oxidation of rMb(CoII(TDHC)) with K3[Fe(CN)6]. The cyanide-coordinated Co(III) species in the latter protein was prepared by ligand exchange of rMb(CoIII(OH2)(TDHC)) with exogenous cyanide upon addition of KCN. The X-ray crystallographic study reveals the hexacoordinated structures of rMb(CoIII(OH)(TDHC)) and rMb(CoIII(CN)(TDHC)) at 1.20 and 1.40 Å resolution, respectively. The 13C NMR chemical shifts of the cyanide in rMb(CoIII(CN)(TDHC)) were determined to be 108.6 and 110.6 ppm. IR measurements show that the cyanide of rMb(CoIII(CN)(TDHC)) has a stretching frequency peak at 2151 cm−1 which is higher than that of cyanocobalamin. The 13C NMR and IR measurements indicate weaker coordination of the cyanide to CoIII(TDHC) relative to cobalamin, a vitamin B12 derivative. Thus, the extent of π-back-donation from the cobalt ion to the cyanide ion is lower in rMb(CoIII(CN)(TDHC)). Furthermore, the pK1/2 values of rMb(CoIII(OH2)(TDHC)) and rMb(CoIII(CN)(TDHC)) were determined by a pH titration experiment to be 3.2 and 5.5, respectively, indicating that the cyanide ligation weakens the Co−N(His93) bond. Theoretical calculations also demonstrate that the axial ligand exchange from water to cyanide elongates the Co−N(axial) bond with a decrease in the bond dissociation energy. Taken together, the cyano-Co(III) tetradehydrocorrin in myoglobin is appropriate for investigation as a structural analogue of methylcobalamin, a key intermediate in methionine synthase reaction.

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accelerated by ca. 105-fold within the enzyme compared to the nonenzymatic reaction.2b,d,3 In order to understand the enzymatic reaction mechanism, it is essential to consider the effects on the electronic and structural properties of the corrin framework as well as the axial ligand.4−11 To evaluate the chemical properties of the corrin framework, two characteristic cobalt corrinoids, di- and tetradehydrocorrin cobalt complexes, have been reported as models of cobalamin.4 The former cobalt complex is capable of forming a stable Co(III)−C bond similar to that of methylcobalamin. The latter cobalt complex (Figure 1b) provides a stable Co(I) species, but the subsequent alkylation reaction does not occur in organic solvents.4 Furthermore, several substituted cobalamins with substitution at the 10position of the corrinoid and cobalt corrole derivatives have also been synthesized to investigate the framework effect using theoretical and experimental methods.5,6 In addition to the

INTRODUCTION Methylcobalamin is a physiologically relevant organometallic cofactor in most living organisms.1 Its precursor, cobalamin, is a cobalt complex with a corrin framework which acts as a monoanionic ligand with four highly saturated pyrrole rings (Figure 1a). The cobalt complex forms an intramolecular coordinate bond with a peripherally attached 6,7-dimethylbenzimidazole (DMB) moiety at the 17-position of the framework in cobalamin-dependent enzymes such as diol dehydratase.1 In contrast, methylcobalamin interacts with a histidine residue as an axial ligand, and the DMB moiety is free from the cobalt ion in methionine synthase.2 The catalytic cycle of the latter enzyme is known to involve two characteristic intermediates, Cob(I)alamin and methylcobalamin. The Co(I) species reacts with N-methyltetrahydrofolate to yield methylcobalamin with an organometallic Co(III)−CH3 bond, and then the enzyme is responsible for catalysis of a transmethylation reaction, converting methylcobalamin to homocysteine to produce methionine.2 It is noted that the reaction via the heterolytic cleavage of the Co(III)−CH3 bond of methylcobalamin is © XXXX American Chemical Society

Received: November 10, 2015

A

DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

and rMb(CoIII(CN)(TDHC), respectively, as models of methylcobalamin in the enzyme matrix (Figure 1b,c).

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EXPERIMENTAL SECTION

Instruments. UV−vis spectral measurements were carried out with a Shimadzu UV-3150 or UV-2550 double-beam spectrophotometer, or a Shimadzu BioSpec-nano spectrometer. 13C NMR spectra were collected on an Avance III (600 MHz) NMR spectrometer. IR spectra were recorded with a JASCO FT/IR-6200 spectrometer using the SL2 demountable cell unit (optical path-length = 0.025 mm, Pier Optics Co., Ltd., Japan) with CaF2 windows. ICP-OES (inductively coupled plasma optical emission spectroscopy) was performed with a Shimadzu ICPS-7510 emission spectrometer. The pH measurements were made with an F-52 Horiba pH meter. Materials. All reagents of the highest guaranteed grade available were obtained from commercial sources and were used as received unless otherwise indicated. Distilled water was demineralized using a Barnstead NANOpure DIamond or Millipore Integral3 apparatus. Synthesis of cobalt tetradehydrocorrin, Co(TDHC), was described in our previous report.10 Native horse heart myoglobin, nMb (SigmaAldrich), was purified with a cation exchange CM-52 cellulose column. The apoprotein was prepared according to Teale’s method.15 The reconstituted protein, rMb(CoII(TDHC)), was obtained by the conventional method.10 A cobalt standard solution for ICP-OES was purchased from Wako Pure Chemical Industries. Preparation of rMb(CoIII(OH2)(TDHC)). Into a solution of rMb(CoII(TDHC))10 (50 μM, 5 mL, 0.25 μmol) in 0.1 M potassium phosphate buffer at pH 7.0 was added a solution of K3[Fe(CN)6] (0.1 M, 0.25 mL, 25 μmol, 100 equiv). After equilibrating at 4 °C under aerobic conditions for 2 h, the protein was purified using a HiTrap desalting column (5 mL, GE Healthcare) with 0.1 M potassium phosphate buffer at pH 7.0. The protein solution was stored in the dark at −80 °C. UV−vis spectral data of the protein are shown in Table 2. Preparation of rMb(CoIII(CN)(TDHC)). Into a solution of rMb(CoII(TDHC)) (50 μM, 5 mL, 0.25 μmol) in 0.1 M potassium phosphate buffer at pH 7.0 were added a solution of K3[Fe(CN)6] (0.1 M, 50 μL, 5 μmol, 20 equiv) and a solution of KCN (50 mM, 50 μL, 2.5 μmol, 10 equiv). After equilibrating at 4 °C under aerobic conditions for 12 h, the protein was purified using a HiTrap desalting column (5 mL, GE Healthcare) with 0.1 M potassium phosphate buffer at pH 7.0. The protein solution was stored in the dark at −80 °C. UV−vis spectral data of the protein are shown in Table 2. ESITOF MS (negative mode, m/z): [M − 7H+]7− calcd for C803H1241CoN215O222S2, 2511.59; found, 2511.62. UV−Vis Spectral Measurements of Co(TDHC) Complexes without the Protein Matrix as a Control Experiment. Into a solution of CoII(TDHC) (10 μM, 20 mL, 0.2 μmol) in 0.1 M potassium phosphate buffer at pH 7.0 was added a solution of K3[Fe(CN)6] (0.1 M, 0.2 mL, 20 μmol, 100 equiv). After equilibrating at 4 °C under aerobic conditions for 2 h, the oxidized Co(III) species, CoIII(TDHC)L2 (L = H2O or OH−), was obtained. Subsequently, into the solution of CoIII(TDHC)L2 (10 μM, 5 mL, 0.05 μmol) was added a solution of imidazole (0.5 M, 10 μL, 5 μmol, 100 equiv) to yield the imidazole-ligated species. Into the solution of CoIII(TDHC)L2 (10 μM, 5 mL, 0.05 μmol) was added a solution of KCN (50 mM, 50 μL, 2.5 μmol, 50 equiv) to yield the dicyano species, CoIII(CN)2(TDHC). The UV−vis spectrum of each species was recorded at 25 °C without any purification processes. The UV−vis spectral data are summarized in Table 2. Autoxidation of rMb(CoII(TDHC)) in the Presence of KCN. Into a solution of rMb(CoII(TDHC)) (50 μM, 1 mL, 0.05 μmol) in 0.1 M potassium phosphate buffer at pH 7.0 was added a solution of KCN (50 mM, 10 μL, 0.5 μmol, 10 equiv). After equilibration at 4 °C under aerobic conditions for 12 h, the UV−vis spectrum of the solution was monitored. Determination of pKa and pK1/2 Values. UV−vis spectra of rMb(Co III (OH 2 )(TDHC)), rMb(Co III (CN)(TDHC)), rMb(CoII(TDHC)), and nMb (10−40 μM) in 0.1 M potassium phosphate

Figure 1. Molecular structures of (a) cobalamin and (b) Co(TDHC), and (c) reaction scheme for oxidation of rMb(CoII(TDHC)) and subsequent ligand exchange with a cyanide ion.

framework effect, various cobalamin derivatives were synthesized to investigate the axial-ligation effect, particularly the relationship between the Co(III)−C and Co(III)−N bond lengths.1d,5b,7 A bulky axial ligand such as DMB is known to cause elongation of the Co(III)−C bond due to upward folding of the corrin ring.1d,8 This axial ligation generally promotes homolytic cleavage of the Co(III)−C bond as a result of electronic stabilization of the produced Co(II) species.1d,8 In contrast, there are few appropriate cobalamin models which replicate the DMB-off/His-on ligation which occurs in the reaction cycle of methionine synthase, where the methyl group bound to the cobalt atom is transferred to homocysteine via the heterolytic Co(III)−C bond cleavage.9,12 To evaluate the effect of the histidine ligation, it is of interest to construct a conjugate model between a cobalt complex and a protein matrix.10,11,13 In our previous work, myoglobin reconstituted with tetradehydrocorrin cobalt(II) complex, rMb(CoII(TDHC)), was prepared to replicate the cobalamin-binding domain of methionine synthase, because the heme pocket of myoglobin possesses a potent histidine (His93) ligand which coordinates to heme in the native protein. The X-ray crystal structure analysis reveals that the His93 residue coordinates to the Co(II) corrinoid, and then reduction of the crystals leads to the tetracoordinated Co(I) corrinoid after deligation of the His93 residue in the heme pocket.10 Furthermore, it has been found that the Co(I) species reacts with methyl iodide to form the Co(III)−CH3 bond and the subsequent intraprotein transmethylation occurs in the heme pocket.4,11 In these transmethylation events as a model reaction of methionine synthase, the theoretical study suggests that ligation and deligation of the axial histidine residue in the myoglobin matrix play important roles in the formation and activation of the Co(III)−CH3 species, respectively.11 However, an X-ray crystal structure of the methylated complex has not been available because the organometallic Co(III) species is more unstable than methylcobalamin11 and rapidly decomposes when subjected to X-ray irradiation.14 Here, we report the structures and physicochemical properties of relatively stable aqua- and cyanoCo(III) complexes of myoglobin, rMb(CoIII(OH2)(TDHC)) B

DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry buffer at 25 °C were measured under conditions of varying pH values which were adjusted by incremental addition of aqueous solutions of 1−12 M HCl or 10 M NaOH. The pH values of the solution were recorded before and after each spectroscopic measurement. The pKa and pK1/2 values were determined from the data which are fitted to the following Henderson−Hasselbach equation (eq 1) for a one proton transfer process16

Table 1. Data Collection and Refinement Statistics for the Reconstituted Myoglobins rMb(CoIII(OH) (TDHC)) Data Collection PDB ID X-ray source detector wavelength (Å) resolution (Å) (outer shell) space group unit cell params (Å, deg) total reflns unique reflns completeness (%) Rsyma I/σ(I) Refinement resolution (Å) no. of reflns Rcryst/Rfree (%) mean B-factor (Å2) no. of non-H atoms bond lengths (Å)/ angles (deg)

(pH − pK )

Z=

A acid + A neutral × 10

1 + 10(pH − pK )

rMb(CoIII(CN) (TDHC))

(1)

where Z is absorbance at a certain pH, and Aacid and Aneutral are absorbances of the acid and neutral forms, respectively. Protein Crystallization. Crystallization of rMb(CoII(TDHC)) was performed at 25 °C using the hanging-drop vapor-diffusion method.10 The protein drops were prepared by mixing of 5 μL of the protein solution (10 mg/mL) in 0.1 M Tris-HCl buffer at pH 7.4 with 5 μL of a reservoir solution at pH 7.4 containing 3.4 M ammonium sulfate, 0.1 M Tris-HCl, and 10% trehalose. The obtained crystals were soaked in the reservoir solution containing K3[Fe(CN)6] (15.2 mM) to prepare the crystals of the aqua-Co(III) species. A color change from dark red to green was observed within 10 days, and the crystal was then fished and flash-cooled in a stream of N2 gas at 100 K. To prepare the crystals of the cyano-Co(III) species, the crystals of rMb(CoII(TDHC)) were soaked in the reservoir solution containing 5 mM KCN and 117 mM K3[Fe(CN)6]. A color change from dark red to green was observed within 20 min, and the crystal was then fished and flash-cooled in a stream of N2 gas. Crystal Structure Determination and Refinement. X-ray diffraction data were collected at beamline BL44XU of SPring-8 (Harima, Japan). The data were processed with the program HKL2000.17a The structures were determined by the molecular replacement method using the program PHASER17b from the CCP4 program suite.17c A crystal structure of horse heart myoglobin (Protein Data Bank (PDB) ID: 3WFT) was used as the search model. Refinement was carried out using the program REFMAC.17d The structures were visualized and modified using the program COOT.17e Since the electron density of Co(TDHC) indicates that two enantiomers (1R,19R)- and (1S,19S)-TDHCs of Co(TDHC) exist in the heme pocket, we assigned approximate RR:SS occupancy with a ratio of 65:35 in both proteins. Data collection and refinement statistics are summarized in Table 1. NMR Measurements of rMb(CoIII(CN)(TDHC)). Into a solution of rMb(CoII(TDHC)) (0.55 mM, 3 mL, 1.65 μmol) in 0.1 M potassium phosphate buffer at pH 7.0 was added a solution of normal or 13C-enriched KCN (0.1 M, 33 μL, 3.3 μmol, 2 equiv), and then, the mixture was equilibrated at 4 °C under aerobic conditions for 30 min. After concentration to 0.8 mL using an Amicon Ultra-4 centrifugal filter (10 kDa) (GE Healthcare), the protein solution was passed through a HiTrap desalting column (GE Healthcare) equilibrated with 0.1 M potassium phosphate buffer at pH 7.0. After addition of D2O (final concentration of D2O: 10% v/v), the solution was concentrated to 0.4 mL and transferred to an NMR microtube (Shigemi Co., Ltd., Hachioji, Japan). The 13C NMR measurements of the proteins were carried out at 5 °C over 2 days (65 536 scans). As the control experiments of the reconstituted proteins, CoIII(13CN)2(TDHC) was prepared by autoxidation of CoII(TDHC) (2 mM) in the same buffer containing K13CN (4 mM) under aerobic conditions. FT-IR measurements of rMb(CoIII(CN)(TDHC)). Each solution containing 13C-enriched or normal cyanide bound to the cobalt complex (ca. 6 mM) in 0.1 M potassium phosphate buffer at pH 7.0 was prepared. A series of FT-IR spectra were accumulated (50 scans) at 25 °C with 4.0 cm−1 resolution. Relaxation Analysis of Cyanide Binding. In general, it is difficult to determine the affinity of cyanide for aqua-Co(III) complexes, because the ligand-exchange process is too slow to detect by conventional titrimetric measurements at 25 °C, pH 7.0, in the presence of KCN (10 μM).18 Thus, the binding constant of cyanide was determined by a relaxation analysis (Figure S1).18 Into a solution

5AZR SPring-8 BL44XU Rayonix MX225HE 0.900 00 50−1.20 (1.24−1.20)

5AZQ SPring-8 BL44XU Rayonix MX225HE 0.900 00 50−1.40 (1.45−1.40)

P21 a = 34.79, b = 28.84, c = 63.32, β = 106.17 136 063 37 167 97.6 (97.5) 5.8 (42.8) 20.4 (3.1)

P21 a = 34.77, b = 28.81, c = 63.26, β = 105.64 85 185 23 585 97.8 (95.5) 5.6 (37.6) 22.6 (2.4)

50−1.20 35 357 12.1/16.8 17.3 1476 0.033/3.248

50−1.40 22 308 12.8/18.9 18.8 1456 0.027/3.070

Rsym = ∑hkl∑i|Ii(hkl) − ⟨I(hkl)⟩|/∑hkl∑iIi(hkl), where Ii(hkl) is the value of the ith measurement of the intensity of a reflection, ⟨I(hkl)⟩ is the mean value of the intensity of that reflection, and the summation is over all measurements.

a

of rMb(CoIII(OH2)(TDHC)) (10 μM) in 0.1 M potassium phosphate buffer at pH 7.0 was added a solution of 0.20 or 0.10 M KCN in the same buffer to the desired final concentrations of KCN (0.10, 0.50, 1.0, 1.5, and 2.0 mM), and then UV−vis spectra were recorded at 25 °C for 20 min. The concentrations of the cyanide ion were determined using the following Henderson−Hasselbach equation (eq 2)

⎛ [HCN] ⎞ ⎛ C0 − [CN−] ⎞ pH = pK a − log⎜ p K log = − ⎟ ⎜ ⎟ a ⎝ [CN−] ⎠ ⎝ [CN−] ⎠

(2)

where the pH and pKa values are 7.0 and 9.2 at 25 °C, respectively; C0 is the initial concentration of KCN. The spectral changes occurring over 20 min upon addition of KCN were observed. The transient relaxation kinetics are represented by the following equation (eq 3) 19

kon

rMb(CoIII(H 2O)(TDHC)) + L ⎯⇀ rMb(CoIII(L)(TDHC)) ↽⎯⎯⎯ k off

(3) where L denotes an exogenous ligand; kon and koff represent rate constants for the association and dissociation of L, respectively. The observed rate constant kobs is given by the following equation (eq 4).18 kobs = koff + kon[L]

(4)

The binding constant of cyanide for rMb(CoIII(H2O)(TDHC)) was determined by the following equation (eq 5)

K = kon /koff

(5)

Computational Chemistry. We used the Becke−Perdew (BP86)20 method implemented in the Gaussian 09 program. For all atoms, the 6-31G(d) basis set was used. This BP86/6-31G(d) level of theory serves as an appropriate platform to address the structural, electronic, and spectroscopic properties of cobalamin cofactors.21 All calculations were carried out in the gas phase. As truncated models of Co(TDHC) and cobalamin, we used Co(TDHC′) and Co(corrin), C

DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry respectively, where all of the peripheral side chains in TDHC and corrin are replaced with hydrogen atoms for DFT calculations.11 The bond dissociation energies (BDEs) of the Co−N(Im) bonds of the Im-coordinated Co(III) complexes are defined by the following equation (eq 6) III

III

BDE = E(Co (L)) + E(Im) − E(Co (Im)(L))

Table 2. UV−Vis Spectral Absorption Data in 0.1 M Potassium Phosphate Buffer at pH 7.0 at 25 °C protein or complex rMb(CoIII(OH2)(TDHC)) rMb(CoIII(CN)(TDHC)) rMb(CoII(TDHC))b CoIII(TDHC)L2c CoIII(TDHC)L2 + Imd CoIII(CN)2(TDHC)

(6)

where E(X) is the energy of the optimized structure of X; L and Im are exogenous axial ligand and imidazole, respectively; and CoIII(L) and CoIII(Im)(L) are Im-free and Im-coordinated Co(III) complexes which are coordinated by L, respectively.

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λmax (nm) [εa (mM−1 cm−1)] 629 652 510 629 629 671

[10.0], 686 [19.6] [10.2], 716 [16.7] [16.4] [11.0] [9.1], 677 [11.7] [8.0], 740 [11.6]

a

The molar coefficient was determined from ICP-OES measurements. Reference 10. cL = H2O or OH−. dCoIII(TDHC) (5.2 μM) in 0.1 M phosphate buffer containing 1 mM of Im. b

RESULTS AND DISCUSSION Preparation of rMb(CoIII(OH2)(TDHC)) by Oxidation of rMb(CoII(TDHC)). Addition of K3[Fe(CN)6] to a solution of rMb(CoII(TDHC)) in 0.1 M phosphate buffer at pH 7.0 leads to a new set of absorption maxima (λmax) at 657 and 687 nm with disappearance of the 510 nm absorption (Figure 2a).

Figure 3. (a) UV−vis spectral changes occurring with increasing pH (pH 7−9) at 25 °C. (b) The pH titration curve of the absorbance at 686 nm, which is the absorption maximum of rMb(CoIII(OH2)(TDHC)).

Figure 2. (a) UV−vis absorption spectra of rMb(CoII(TDHC)) (gray dashed line), rMb(CoIII(OH2)(TDHC)) (blue solid line), and rMb(CoIII(CN)(TDHC)) (red solid line). (b) UV−vis absorption spectra of CoIII(TDHC)L2 (L = H2O or OH−) (black line), CoIII(TDHC)L2 in the presence of Im (light blue line), and CoIII(CN)2(TDHC) (pink line). Conditions: 0.1 M potassium phosphate buffer at pH 7.0 at 25 °C.

rMb(CoIII(OH 2)(TDHC)) ⇌ rMb(CoIII(OH)(TDHC)) + H+

(7)

A pKa value of 7.5 for the coordinated water molecule in rMb(Co(OH2)(TDHC)) was determined from data obtained in a pH titration experiment (Figure 3b). This pKa value is similar to that of base-on aquacobalamin (pKa = 7.8) and higher than that of base-off diaquacobalamin (pKa = 6.0), where the base-on and base-off transitions represent the transitions of the intramolecular DMB-ligated and H2O-ligated forms in the cobalamin molecule, respectively.22 This finding suggests that the His93 residue coordinates to CoIII(TDHC) in myoglobin (His-on) under conditions of neutral pH. Ligand Exchange to Obtain the Cyano-Co(III) Complex. The coordinated water molecule in rMb(CoIII(OH2)(TDHC)) is exchangeable with a cyanide ion upon addition of a solution of KCN, giving red-shifted absorption maxima at 652 and 716 nm (Figure 2a and Table 2). These maxima are different from those of the bare CoIII(CN)2(TDHC) complex, which are consistent with those of the reported Co(III) tetradehydrocorrin dicyano-complex in CH2Cl2.4d These results support the formation of a monocyano-adduct of CoIII(TDHC) with His93 coordination in the heme pocket of myoglobin,

These new absorption bands have been attributed to an intermolecular charge transfer transition for the cobalt−axial ligand bond of the CoIII(TDHC) species.4d As a reference experiment, we also monitored the UV−vis spectral change of the bare CoII(TDHC) complex upon addition of K3[Fe(CN)6] and imidazole (Im) as an axial ligand (Figure 2b). The obtained spectrum of the Im-ligated CoIII(TDHC) species is generally consistent with that of CoIII(TDHC) in the protein (Table 2), indicating that CoII(TDHC) is oxidized by K3[Fe(CN)6] to form the Co(III) species, rMb(CoIII(OH2)(TDHC)), coordinated by the His93 residue in the heme pocket of myoglobin at pH 7.0. Increasing the pH value of the oxidized protein solution induces spectral changes with isosbestic points (Figure 3a), which are derived from deprotonation of a water molecule coordinated to the cobalt atom in the reconstituted myoglobin (eq 7). D

DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry rMb(CoIII(CN)(TDHC)), as characterized by ESI-TOF MS. The affinity of the cyanide for rMb(CoIII(OH2)(TDHC)) was determined by relaxation analysis18 and indicated a K value of 8.9 × 105 M−1 at pH 7.0 (Figure S1), which is lower than that of CNCbl (≥1012 M−1).22 Interestingly, rMb(CoIII(CN)(TDHC)) can also be obtained by autoxidation of the Co(II) species in the reconstituted protein upon addition of an excess amount of cyanide (10 equiv) under aerobic conditions, whereas similar oxidation does not occur in the absence of cyanide. The oxidation appears to be initiated by coordination of cyanide to the Co(II) species, which would lead to a negative shift of the Co(II)/Co(III) redox potential of Co(TDHC). Crystal Structures of rMb(CoIII(OH)(TDHC)) and rMb(CoIII(CN)(TDHC)). The crystal of myoglobin with the Co(III) species was obtained by soaking the crystal of rMb(CoII(TDHC))10 in a reservoir solution containing K3[Fe(CN)6] (15.2 mM) at pH 7.4. At this pH, approximately half of the coordinated water molecules in rMb(CoIII(OH2)(TDHC)) are deprotonated to rMb(CoIII(OH)(TDHC)) at room temperature (vide supra). In addition, the pH value of the reservoir solution containing 0.1 M Tris-HCl buffer is found to increase at low temperature (Figure S2), suggesting that the water molecule coordinated to CoIII (TDHC) will be deprotonated to form rMb(CoIII(OH)(TDHC)) under the conditions used for data collection at 100 K. The color of the rMb(CoIII(OH)(TDHC)) crystal changes from green to deep red after X-ray irradiation, indicating that the Co(III) species is reduced to the Co(II) species by the generated photoelectron and/or hydroxyl radical in the protein.14 The X-ray crystal structure of the reconstituted protein was determined and refined at 1.20 Å resolution (Figure 4a). The cofactor is bound in the heme pocket by Co− N(His93) ligation. The electron density map clearly shows two oxygen atoms above the cobalt atom of Co(TDHC). Each oxygen occupancy is assigned to be 50%, and the distances of Co−O1 and Co−

O2 are 1.86 and 3.01 Å, respectively (Figure 5a). These results suggest that, under X-ray irradiation, the hexacoordinated

Figure 5. (a) Electron density map of the cofactor of rMb(CoIII(OH)(TDHC)). The 2Fo − Fc electron density is shown as a blue mesh for the cofactor and a red mesh for the oxygen atoms above the cobalt atom (contoured at 1.0σ). (b) Plausible scheme for the reduction of rMb(CoIII(OH)(TDHC)) to rMb(CoII(TDHC)) which occurs upon X-ray irradiation.

CoIII(OH)(TDHC)−His93 complex is partially reduced to form the pentacoordinated CoII(TDHC)−His93 complex in the heme pocket. After cleavage of the Co−OH bond of rMb(CoIII(OH)(TDHC)), it appears that the released hydroxide accepts the proton bound to the Nε2 atom of the His64 residue (Figure 5b). The produced water molecule is located in the heme pocket and participates in hydrogen bonding with the Nε2 atom of the His64 residue with an O2− Nε2 distance of 2.60 Å. Furthermore, the crystal structure reveals that the Co−O bond length of rMb(CoIII(OH)(TDHC)) is shorter than the Fe−O bond length of native met-aquo myoglobin and the Co−O bond length of myoglobin reconstituted with cobalt protoporphyrin IX, rMb(CoIII(OH2)(PP)). Furthermore, the Co−O bond length of rMb(CoIII(OH)(TDHC)) is much shorter than that of aquacobalamin, H2OCbl,23a and is also shorter than that of hydroxocobalamin, OHCbl (Table 3).7c The relatively short Co−O bond length (1.86 Å) indicates that the water molecule coordinated to the cobalt atom is deprotonated to form the hydroxide ion observed in the crystal structure (Table 3). We also determined the X-ray crystal structure of rMb(CoIII(CN)(TDHC)) at 1.40 Å resolution (Figure 4b). In this case, the X-ray irradiation-induced reduction was not observed. The crystal structure shows the hexacoordinated CoIII(CN)(TDHC) complex bound in the heme pocket by Co− N(His93) ligation. To the best of our knowledge, this is the first known instance of determination of 3D-structures of a hexacoordinated Co(III) tetradehydrocorrin complex with two different ligands at the axial positions.4d Table 3 provides a comparison of several bond distances between axial ligands and metal cofactors. The Co−N(His93) bonds of the reconstituted myoglobins are longer than the Co− N(DMB) bonds of the corresponding cobalamins, although the substitution of DMB of cyanocobalamin (CNCbl, dCo−N(DMB) = 2.04 Å) with Im is known to provide a shorter Co−N bond (1.97 Å).7e,24 The oxidation of rMb(CoII(TDHC)) provides a shorter Co−N(His93) bond in rMb(CoIII(OH)(TDHC))

Figure 4. Crystal structures of (a) rMb(CoIII(OH)(TDHC)) and (b) rMb(CoIII(CN)(TDHC)). The whole protein structures (left), and the structures of cofactors and axial ligands (right), are shown. Each cofactor consists of two enantiomers, (1R,19R)-TDHC and (1S,19S)TDHC, with a ratio of 65:35 due to two chiral centers at the C1- and C19-positions of the corrinoid framework.10 E

DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Table 3. Bond Lengths of Axial Coordination for Co(TDHC)s or Cobalamins protein or complex

M−Xa (Å)

M−N(axial ligand)b (Å)

rMb(CoIII(OH)(TDHC))c rMb(CoIII(CN)(TDHC))c rMb(CoII(TDHC))d nMb(FeIII(OH2)(PP))e rMb(CoIII(OH2)(PP))f H2OCblg OHCblh CNCbli CNCbl−Imj Cob(II)alamink

1.86 2.09

2.09 2.13 2.17 2.26 2.15 1.93 1.99 2.04 1.97 2.13

2.29 2.46 1.95 1.92 1.89 1.86

Figure 6. 13C NMR (150 MHz) spectra of rMb(CoIII(CN)(TDHC)). (a) The samples were prepared with (a) 13C-enriched KCN and (b) nonenriched KCN, respectively. (c) The differential spectrum of a − b. Conditions: [protein] = 5 mM in 0.1 M potassium phosphate buffer at pH 7.0 containing D2O (10%) at 5 °C.

X is the sixth ligand: X = OH−, CN−, or H2O. bDistance between the Co−N(His93) and Co−N(DMB) bonds for myoglobin and cobalamin, respectively. cThis work. dReference 10. PDB ID: 3WFT. e Reference 23b. PDB ID: 1YMb. fReference 23c. PDB ID: 2O5T. g Aquacobalamin, ref 23a. hHydroxocobalamin, ref 7c. iCyanocobalamin (CNCbl), ref 7a. jDMB of CNCbl was substituted with Im, ref 7e. k Pentacoordinated Co(II) species of cobalamin ref 7d. a

Table 4. 13C NMR Chemical Shifts of Cyano-Cobalt Corrinoids

(2.17 Å), which is consistent with the fact that the bond length of OHCbl (1.99 Å) is shorter than that of Cob(II)alamin (2.09 Å). Furthermore, the replacement of hydroxide in rMb(CoIII(OH)(TDHC)) (dCo−N(His93) = 2.09 Å) with cyanide elongates the Co−N(His93) bond in rMb(CoIII(CN)(TDHC)) (2.13 Å). This also occurs during the conversion from OHCbl to CNCbl. Next, comparing the structures of the three cyano-Co(III) complexes listed in Table 3, we found that the Co−N(axial ligand) bond elongates with increasing length of the Co−CN bonds in the order CNCbl-Im < CNCbl < rMb(CoIII(CN)(TDHC)). These cyano-Co(III) complexes exhibit an inverse trans-effect-like property on both axial bond lengths.7 Moreover, the polypeptide Cα atoms of rMb(CoIII(OH)(TDHC)) and rMb(CoIII(CN)(TDHC)) are superimposable on those of nMb with root-mean-square (RMS) deviation values of 0.225 and 0.264 Å, respectively. The oxidation of rMb(CoII(TDHC)) and the subsequent ligand exchange from hydroxide to cyanide do not have a significant influence on structural changes of the protein matrix, although the reduction of the Co(II) species provides a His-off state via flipping of the His93 residue with a significantly high B-factor.10 However, the ligation of cyanide provides slightly higher B-factor values of the His93 residue of rMb(CoIII(CN)(TDHC)) than those of rMb(CoIII(OH)(TDHC)) and rMb(CoII(TDHC)) (Figure S4). This suggests that the Co−N(His93) bond slightly weakens upon binding of cyanide. NMR Spectroscopy. The 13C NMR spectra of rMb(CoIII(CN)(TDHC)) are shown in Figure 6, in which Figure 6a was obtained from a sample with 13C-enriched KCN. The differential spectrum gives two peaks at 108.4 and 110.6 ppm assigned as 13C-enriched cyanide bound to the cobalt ion in the protein matrix (Figure 6c and Figure S5), because of the mixture of the two cofactor enantiomers in the asymmetric protein matrix (vide supra). A ratio of the areas of these peaks was determined to be 6:4 (Figure S5d), which is similar to the ratio of the enantiomers of Co(R,R-TDHC) and Co(S,STDHC) in the protein matrix (65:35) estimated by crystallography. Chemical shifts of the cyanide peak in cyano-cobalt corrinoids are listed in Table 4. In comparison with

protein or complex

δ (ppm)

rMb(CoIII(CN)(TDHC))a

108.4 110.6 121.4 111.8 112.7 140.2

CNCbl(base-on)b CNCbl(base-off)b Co(CN)2(TDHC)a (CN)2Cblb,c a

This work. bReference 25. cDicyanocobalamin.

dicyanocobalamin, (CN)2Cbl, CNCbl(base-on), and Co(CN)2(TDHC), the 13C-cyanide peak of rMb(CoIII(CN) (TDHC)) undergoes an upfield shift (Figure S6). The similar upfield shift of the cyanide peak is seen in CNCbl(base-off). Taken together, these findings suggest that the weaker σdonation from the cyanide ion to the cobalt ion relative to CNCbl(base-on) is due to the longer Co−CN bond of rMb(CoIII(CN)(TDHC)) than that of CNCbl. IR Spectroscopy. IR spectra of rMb(CoIII(13CN)(TDHC)) and rMb(CoIII(12CN)(TDHC)) are shown in Figure 7. The 13 CN/12CN-differential spectrum clearly shows the typical isotope shift.26 The stretching frequency of the cyanide ion in

Figure 7. IR spectra of rMb(CoIII(CN)(TDHC)) prepared with (a) K13CN and (b) K12CN. (c) The differential spectrum of a − b. Conditions: [protein] = 6 mM in 0.1 M potassium phosphate buffer at pH 7.0 at 25 °C. F

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Inorganic Chemistry rMb(CoIII(12CN)(TDHC)) which is assigned at 2151 cm−1 is higher than that of both the mono- and dicyanocobalamins and Co(CN)2(TDHC) (Table 5). The higher stretching frequency

Table 6. pK1/2 Values for Reconstituted and Native Myoglobins and the pKbase‑off Values for Cobalamins protein or complex III

rMb(Co (OH2)(TDHC)) rMb(CoIII(CN)(TDHC))a rMb(CoII(TDHC))a nMb(FeIII(OH2)(PP))a nMb(FeIII(CN)(PP))b H2OCblc,d CNCblc methylcobalaminc

Table 5. Cyanide Stretching Frequencies in Cyanocorrinoids wavenumber (cm−1)

protein or complex III

rMb(Co (CN)(TDHC)) Co(CN)2(TDHC)b CNCblc (CN)2Cblc,d a

a

pK1/2 or pKbase‑off a

2151 2128 2137 2119

This work. bref 27a. cref 27b. dDicyanocobalamin.

3.2 5.5 5.0 5.0 5.3 −2.4 0.1 2.5

a This work. bReference 28. cReference 21. dAquacobalamin. In the following graphic, X is the 6th ligand: X = OH−, CN−, or CH3−.

III

of the cyanide ion in rMb(Co (CN)(TDHC)) is due to weak π-back-donation from the cobalt atom to the cyanide ion,27 indicating that the Co−CN bond is weakened relative to that of CNCbl. These findings are supported by the observation that the Co−CN bond in rMb(CoIII(CN)(TDHC)) is longer than that of CNCbl, as revealed by X-ray crystallography (vide supra). Determination of pK1/2 Values for Reconstituted Myoglobins. The pK1/2 value for each reconstituted protein, which represents the pH value corresponding to a 50% loss of a cofactor from the myoglobin matrix via the cleavage of the metal−N(His93) bond, was determined by fitting the titration curve (Figure 8 and Table 6). From the UV−vis spectral changes of rMb(CoIII(OH2)(TDHC)) and rMb(CoII(TDHC)) which occur when the pH decreases, the pK1/2 values were determined to be 3.2 and 5.5, respectively. rMb(CoIII(OH2)(TDHC)) has the lower pK1/2 value than rMb(CoII(TDHC)), indicating that the oxidation of the Co(II) species enhances the Co−N(His93) bond strength because the Co(III) species has

higher Lewis acidity than the Co(II) and Fe(III) species. In contrast, the pK1/2 value of rMb(CoIII(CN)(TDHC)) is 2.3 pH units higher than that of rMb(CoIII(OH2)(TDHC)) (Figure 8c), while the pK1/2 values of nMb(FeIII(OH2)(PP)) and nMb(FeIII(CN)(PP)) are slightly different (0.3 pH units). Table 6 also demonstrates that, in the case of cobalamins, an increase in the electron-donating ability of an axial ligand increases the pKbase‑off values (see the footnotes under Table 6),7f,22 indicating that the strong electron-donating cyanide ligand weakens the Co−N(His93) bond of rMb(CoIII(CN)(TDHC)). This finding is supported by the IR and X-ray structure data which indicate elongation of the Co−N(His93) bond. Theoretical Calculations for the Co(III) Species of the TDHC and Corrin Frameworks. To characterize the weakening of the Co−N(His93) bond of the Co(III) species by axial coordination of cyanide, we computed the BDE with density functional theory (DFT) calculations (Table 7). As truncated models of Co(TDHC) and cobalamin, we used Co(TDHC′) and Co(corrin), where all of the peripheral side chains are replaced with hydrogen atoms for the DFT calculations (Figure S7). Im was used as a simplified model of the axial His93 residue. The BDEs of the Co−N(Im) bonds of CoIII(OH2)(Im)(TDHC′) and CoIII(CN)(Im)(TDHC′) were calculated to be 47.9 and 20.5 kcal/mol, respectively, indicating that ligation of cyanide ion decreases the BDE of the Co−N(Im) bond. This is also supported by the higher pK1/2 value of rMb(CoIII(CN)(TDHC)) (Table 6). Furthermore, it is noted that the methylation of Co(Im)(TDHC′) significantly reduces the BDE value of the Co−N(Im) bond (14.3 kcal/ mol). The data listed in Table 7 indicate that the Co−N(Im) bond of Co(Im)(TDHC′)(X) lengthens as the BDE value of the Co−N(Im) bond decreases. The elongation of the bond length of the Co−N(Im) which occurs upon binding of cyanide is supported by the X-ray crystal structure data.7,29,30 In the case of rMb(CoIII(CH3)(TDHC)), the observed elongation of the Co−N(His93) bond length predicts the deligation of His93, which promotes the transmethylation in the myoglobin matrix.11 Such ligation and deligation events should also be significant for the native enzyme to regulate the reactivity of methylcobalamin.

Figure 8. UV−vis spectral changes of (a) rMb(CoIII(OH2)(TDHC)) and (b) rMb(CoIII(CN)(TDHC)) occurring with decreasing pH at 25 °C. (c) The pH titration curves of the absorbance at 686 nm for rMb(CoIII(OH2)(TDHC)) (blue ▲) and at 716 nm for rMb(CoIII(CN)(TDHC)) (red ●). G

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Table 7. Bond Dissociation Energy (BDE) of the Co−N(Im) Bond and Bond Lengths of Co−X and Co−N(Im) Bonds in CoIII(TDHC′)(Im)(X)a,b bond length (Å)

a

cobalt complex

BDE of Co−N(Im) (kcal/mol)

Co−X

CoIII(OH2)(Im)(TDHC′)c CoIII(CN)(Im)(TDHC′)c CoIII(CH3)(Im)(TDHC′)d

47.9 20.5 14.3

2.08 1.85 1.98

b

Co−N(Im) 1.90 2.08 2.21

At the BP86/6-31G(d) level of theory bX is the sixth ligand: X = OH2, CN−, or CH3−. cThis work. dReference 11.

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Notes

CONCLUSION This work demonstrates that myoglobins reconstituted with aqua- and cyano-cobalt tetradehydrocorrins are appropriate for investigation as simplified cobalamin models. It is of particular interest to evaluate the binding characteristics of axial ligands for Co(III) corrinoids. 13C NMR and IR studies of rMb(CoIII(CN)(TDHC)) indicate that the cyanide weakly interacts with CoIII(TDHC). The σ-donation from the cyanide ion to CoIII(TDHC) is relatively weaker than that of cyanide-binding cobalamin. Furthermore, the large pK1/2 value of rMb(CoIII(CN)(TDHC)) suggests that cyanide binding weakens the Co−N(His93) bond strength. In addition, the X-ray crystal structure analysis reveals that the Co−N(His93) bond length is slightly elongated. The DFT calculations also clearly support the unique axial ligation; the Co−N(Im) bond is elongated, and the BDE of Co−N(Im) is significantly decreased upon replacement of H2O with cyanide as an axial ligand. Given these findings, it appears that the cyanide-binding species in Co(TDHC) is a transient structural model of methylcobalamin which promotes methyl group transfer to homocysteine in methionine synthase. In the enzyme-catalyzed reaction, it is likely that axial histidine ligation modulates both the stabilization and activation of the methyl group bound to cobalamin. Here, it is found that cyanide binding clearly has a significant influence on His93 ligation. Mechanistic investigations of the methyl transfer reaction in the protein are now in progress.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research provided by JSPS and MEXT (24655051, 15H00944, 15H05804, 22105013, 26104523, 15K18487), the JSPS Japanese-German Graduate Externship. We thank the beamline staff of SPring-8 for their support (proposal nos. 2012B6745 and 2013B1148). Y.M. acknowledges support from the JSPS Research Fellowship for Young Scientists (14J00790).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02598. Relaxation analysis of cyanide binding; a correlation of the pH values of the reservoir solution and temperature; the superimposed crystal structures of nMb and reconstituted proteins rMb(CoIII(OH)(TDHC)) and rMb(CoIII(CN)(TDHC)); the schematic representation for B-factors of the reconstituted proteins; NMR spectra of rMb(CoIII(CN)(TDHC)) and CoIII(CN)2(TDHC); the truncated models for the DFT calculations; schematic representations for the cyano complexes; a table of DFT-optimized bond lengths, atomic charges, stretching frequencies, and BDEs of CoIII(CN)(Im)(TDHC′) and CoIII(CN)(Im)(corrin); and the coordinates of the DFT-optimized structures of CoIII(Im)(TDHC′)(X) complexes and CoIII(CN)(corrin)(Im) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. H

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DOI: 10.1021/acs.inorgchem.5b02598 Inorg. Chem. XXXX, XXX, XXX−XXX