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Chapter 9
Bis-Peroxo-Oxovanadium(V) Complexes of Histidine-Containing Peptides as Models for Vanadium Halo-Peroxidases 1
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J. A. Guevara-García , N. Barba-Behrens , R. Contreras , and G. Mendoza-Díaz 1
Centro de Química-Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, P.O. Box 1613, Puebla 72000, México División de Estudios de Posgrado-Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, D.F. 04510, México Departamento de Química, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, P. O. Box 14-740, México, D.F. 07000, México Facultad de Química, Universidad de Guanajuato, Guanajuato 36050, México 2
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Reaction products of V O /H O with histidine-containing-peptides and NH OH with formula NH [VO(O ) (P)] [where P= gly-L-his (1), gly-gly-L-his (2) and gly-L-his (3)] are isolated and characterized by IR, H, solution C and V and solid C NMR. Solution H and C NMR spectra for 2 and 3 show three sets of signals for each peptide product, which agree with the V NMR features; an HETCOR ex periment enable to propose that compounds 1-3 correspond to the pep tide complexes coordinated through the imidazole. Solid CNMR spectra is consistent with solution C NMR. These complexes may be models for the enzyme vanadium chloroperoxidase, since the vana dium-histidine bond is the only anchor of the prostetic group. The structural and catalytic role of histidine in the active center of a number of enzymes is well known (1-3), as for vanadium haloperoxidases (VHPO) this role has just began to be disclosed (4,5). The first vanadium haloperoxidase isolated was the vanadium bromoperoxidase (VBrPO) (6). EXAFS studies showed that at least one histidine was in the coordination sphere of vanadium(V) (7), while EPR studies suggested that an imidazolic proton from histidine was involved (8). The X-ray structure of the azide complex of the enzyme vanadium chloroperoxidase (VC1PO) (9) showed that the vanadium(V) ion was coordinated by an axial nitrogen from a histidine with a second histidine hydrogen-bonded to the azide. The position of these histidine residues resembles those found for the proximal and distal histidine residues in hemoperoxidases and catalases. It has been suggested that the proximal histidine regulates the redox chemistry of the metal ion while the distal one plays a role in the acid-base catalysis of the substrate (2,3). Systems involving peroxo and bis-peroxo vanadates with small peptides in aqueous solution have been extensively studied by V N M R (10-13). The formation constants determined for various vanadium/ligand pairs indicate the possibility to obtain stable complexes. The crystal structure for the complex (NEt )[V0(0 )(glygly)] (14) and for the compound [V0(0NH ) (glygly)] (15), reported by Tracey et al, confirmed this prediction. A bidentade coordination mode is shown for the glycylglycine in the last complex, with the terminal amine bonded to the vanadium ion in the same plane as the two hydroxylamine ligands and the oxygen 2
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©1998 American Chemical Society In Vanadium Compounds; Tracey, Alan S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
127 atom of the amide group coordinated in the apical position. This coordination mode is also observed for other bis-peroxovanadate complexes (16,17) with ligands such as oxalic acid (18,19), picolinate(20) and phenantroline (21) (Figure \). Polydentade histidine-containing peptides could be better ligands than histidine itself, due to the chelate effect and to the growth of the aminoacids chain which may enhance the stability of the complex through the increase of the hydrogen bonding network. Herein we report a study of the reaction of three peptides gly-L-his (1), glygly-L-his (2), gly-L-his-gly (3), with V 0 , H 0 and ammonia in aqueous solution and the spectroscopic characterization of the products by IR, U V , VIS, *H, C and V solution N M R and C solid N M R . 2
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Experimental Section Materials. Absolute EtOH was purchased from J. T. Baker Co. V 0 , H 0 (30%) and NH4VO3 from Merck Chemical Co.; V O C l from Strem Chemical Co.; gly-L-his from Fluka Chemical Co.; gly-gly-L-his and gly-L-his-gly ICN Chemical Co. Water was destilled and deionized. 2
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Physical Measurements. UV-VIS and diffuse reflectance spectra were recorded with a Cary 5E UV-Vis-near infrared spectrophotometer in the range 250-2500 nm. IR spectra were recorded on a Nicolet FT-IR 740 spectrophotometer using nujol mulls in the range 4000-400 cm" and polyethylene pellets in the range 700-70 cm' . Elemental analyses for C, H, and N were performed in a Fisons Eager 200. Vanadium analyses by plasma atomic absorption were performed at the Universidad Autonoma de Hidalgo. Magnetic susceptibilities of powder samples were measured at it with a Johnson Matthey Gouy balance using the Evan's method for solid samples. Conductivity measurements were done in aqueous solution in an Orion 140 conductivity cell. 1
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NMR Studies. Solid CP/MAS C N M R spectra were recorded at 75.429 MHz, in a 300 MHz Varian Unity Plus spectrometer using total suppression of lateral bands at ca. 3300 spin speed. The following parameters were used: contact times, 1.5 ms; acquisition time, 0.07 s; spectral width, 30030 Hz; number of transients, 512-700; and pulse delay, 4 s. Solution V N M R spectra were recorded in the same equipment at 78.850 MHz, chemical shifts were referenced to V O C l by using the following parameters: acquisition time, 0.2 s; spectral width, 10 Hz; and number of transients, 64300. The samples were dissolved in a mixture 50/50 of H 0 and D 0 . Solution H and C were recorded at rt with a Jeol GSX-300 M H z in D 0 and in DMSO-d using TMS as reference. 5 1
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Synthesis. The peroxo compounds 1-3 were obtained using the following procedure. The corresponding peptide (2 mmol) was dissolved in H 0 30% (8 ml), V 0 (1 mmol) was added in solid to this solution under constant stirring. After V 0 was dissolved, aqueous ammonia was dropped into the solution up to pH 6. The reaction mixture was stirred until the bubbling stopped. Addition of cold ethanol to the reaction mixture produced a yellow precipitate, the suspension was kept in refrigeration overnight. The suspension was decanted and the solid resuspended with a cold acetone/ethanol mixture (3/1 v/v) and kept in refrigeration, this operation was repeated until the overnatant liquid was clear and colorless. The suspension was decanted and the precipitate dried in a vacuum. The compounds were stable under N . Anal. Cald. for 1 [VO^Mgly-L-his^^^O: C, 24.65, H 4.55, N 14.37, V 12.86. Found: C 23.90; H 4.30; N 14.51; V 13.08. Anal. Cald. for 2 [VO(0 ) (glygly-L-his)]-3H 0: C 24.44, H 4.79, N 15.84, V 10.79. Found: C 24.61; H 4.62, N 15.61, V 10.35. Anal. 2
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In Vanadium Compounds; Tracey, Alan S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
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Figure 1. Examples of peroxo-vanadium compounds structures determined by X ray single crystal studies: Associated pair of anions of [VO(0 )(gly-gly)]" (adapted from ref. 14) (A); Molecular structure of V0(NH 0) (gly-gly) (adapted from ref. 15) (B); Molecular structure of [VO(O ) (5-mtro-l,10-phen)]' (adapted from ref. 21) (C). 2
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In Vanadium Compounds; Tracey, Alan S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
129 Cald. for 3 NH [VO(0 ) (gly-L-his-gly)]-H 0 C 28.34, H 4.97, Ν 18.02, V 10.92. Found: C 28.39, Η 4.89, Ν 17.98, V 11.24. 4
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Results and Discussion
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The V N M R spectra of coordination compounds of imidazole or N-methylimidazole bis-peroxovanadate show a signal at -750 ppm attributed to the nitrogen coordination of the vanadium (12). In the histidine coordination compounds two reports indicate that two signals (-750, -740 ppm) appeared for the reaction products. The first one could be attributed to the imidazole coordination whereas the other one could be due to a chelate with the imidazole and carbonyl groups (12, 22). In other report it was established that the coordination with the C0 " group from gly-peptides gives a signal diXca. -710 ppm (11). The V N M R spectra in D 0 for compound 1 (Table I) showed three signals (Figure 2), one at -752 ppm (90%) attributed to compound la, another one at -742 ppm (10%) attributed to compound lb and a third one at -695 ppm corresponds to free bis-peroxovanadate (10). In the C N M R spectra only one set of signals was ob served attributed to compound la. This compound shows an strong effect on the chemical shift of the imidazole carbon atoms. In the ' H N M R spectrum the signals of the imidazole became broad indicating also the interaction of this ring with the vana dium atom, (Figure 3). A n HETCOR experiment was done to assign the resonances. For compounds 2, the V , N M R spectrum has three signals one at -754 assigned to 2a, another at -737 for 2b and a third one at -714 for 2c. C and *H N M R spectra (Table II) showed three sets of signals in the same ratio as for the V spectrum. For compound 2a the spectrum in D 0 solution shows that the coordination of the bisperoxovanadate is through the imidazole nitrogen of the zwitterionic form of the pep tide. For compounds 3, the V N M R spectrum showed the same resonances but in C N M R a strong effect on the carbon atoms of the chain is detected, which could indi cate that for this molecule an additional interaction through the carbonyl atoms is possible (Table III). For compound la it was possible to obtain the N M R Ή and C spectra in DMSO-d which were compared with those obtained in D 0 solution and for the solid state. A completely different coordination behavior was detected from the spectra in DMSO-d solution. The strong shift to higher frequencies of the signals of the carbon atoms of the aminoacid chain shows coordination of the carbonyl oxygen atoms to the vanadium. We were unable to obtain good N M R spectra in DMSO-d for 2a and 3a because of the strong interaction of this solvent with the compounds. In the C solid state N M R spectrum of compound la compared with that ob tained in DMSO-d could indicate an interaction through the imidazole ring and his tidine carboxylate (Table I). The same effect was observed in the solid state N M R spectrum of 2a (Table II), whereas comparison of the solid state N M R spectrum for 3a and its N M R spectra in D 0 solution (Table III) indicates an additional interaction through the carbonyl groups. The C N M R signals for compounds 2b-3b and compounds 2c-3c indicate analogous coordination modes through imidazole. IR and UV-Vis spectroscopic data for compounds 1 to 3 are displayed in Ta ble IV. The IR spectra of the coordination compounds showed new bands at 950-970 cm" and 870 cm" due to v(V=0) and v(O-O) respectively, characteristic of peroxooxovanadium compounds (23,24). Their reflectance spectra contain a band in the re gion 370-380 nm assigned to the charge transfer band V(V)
