Article Cite This: J. Phys. Chem. A XXXX, XXX, XXX−XXX
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Off to the Races: Comparison of Excited State Dynamics in Vitamin B12 Derivatives Hydroxocobalamin and Aquocobalamin Theodore E. Wiley,† Nicholas A. Miller,† William R. Miller,† Danielle L. Sofferman,‡ Piotr Lodowski,⊥ Megan J. Toda,# Maria Jaworska,⊥ Pawel M. Kozlowski,*,#,∇,◆ and Roseanne J. Sension*,†,§,∥ †
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, United States Applied Physics, University of Michigan, 450 Church Street, Ann Arbor, Michigan 48109-1040, United States § Department of Physics, University of Michigan, 450 Church Street, Ann Arbor, Michigan 48109-1040, United States ∥ Biophysics, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, United States ⊥ Institute of Chemistry, University of Silesia in Katowice, Szkolna 9, PL-40 006 Katowice, Poland # Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, Kentucky 40292, United States ∇ Department of Food Sciences, Medical University of Gdansk, Al. Gen J. Hallera, 107, 80-416 Gdansk, Poland
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‡
S Supporting Information *
ABSTRACT: Ultrafast time-resolved spectroscopy was used to study the photochemistry of hydroxocobalamin (HOCbl) and aquocobalamin (H2OCbl+) in solution. Spectroscopic measurements and TD-DFT simulations provide a consistent picture of the spectroscopy and photochemistry. Excitation of H2OCbl+ results in formation of an excited state followed by rapid internal conversion to the ground state (0.35 ± 0.15 ps) through an S1/S0 seam at a slightly elongated Co−O bond length and a significantly elongated Co−NIm bond length. In contrast, the initial elongation of the axial bonds in HOCbl is followed by contraction to an excited state minimum with bonds slightly shorter than those in the ground state. Internal conversion to the ground state follows on a picosecond time scale (5.3 ± 0.4 ps). For both compounds, photodissociation forming cob(II)alamin and hydroxyl radicals (∼1.5% yield) requires excitation to highly excited states. Dissociation is mediated by competition between internal conversion to the S1 surface and prompt bond cleavage.
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enzymatic catalysis.1−3 Apart from the enzymatic environment, the Co−X (X = C, O, or S) bond can often be cleaved homolytically using light.12−15 In particular, excitation of HOCbl at wavelengths shorter than 300 nm results in cleavage of the C−O bond with the formation of radical species.12 The production of OH radicals is potentially useful in a variety of research areas including DNA cleavage, the Fenton reaction, and photosensitive bioactive reagents.14,15 In light of these applications, it is important to understand the photochemistry of both HOCbl and H2OCbl+. The photolysis of HOCbl has been studied using both timeresolved spectroscopy and time-dependent density functional theory (TD-DFT).12,13,16−18 These studies have provided preliminary insights into the photochemistry of HOCbl. There is no reason, a priori, to think that the mechanisms for photolysis of HOCbl and H2OCbl+ will be identical because H2OCbl+ is a
INTRODUCTION Vitamin B12 and its derivatives, also known as cobalamins, are complex organometallic compounds with various functions dependent upon the nature of the upper axial ligand (Figure 1). Cobalamins feature a cobalt atom that is centered in a corrin ring via coordination to the four nitrogens. An attached 5,6dimethylbenzimidazole (DBI) base supplies the lower axial ligand, although in some cases the DBI base is replaced by histidine or water. Enzymatically active cobalamins include species where the upper axial ligand is 5′-deoxy-5′-adenosyl (Ado) or methyl (Me),1−3 while inactive cobalamins of note include antivitamins,4,5 vitamin B12 (cyanocobalamin CNCbl), hydroxocobalamin (HOCbl, vitamin B12b), and aquocobalamin (H2OCbl+, vitamin B12a). However, HOCbl and H2OCbl+ are the aerobic products following photolysis of MeCbl or AdoCbl and are implicated in the function of some B12-dependent photoreceptors.6−11 Cobalamins exhibit unique chemical properties upon cleavage of the bond to the upper axial ligand. Generally speaking, the Co−C bond can be cleaved via homolysis or heterolysis in © XXXX American Chemical Society
Received: June 26, 2018 Revised: July 22, 2018
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DOI: 10.1021/acs.jpca.8b06103 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A
between 535 and 575 nm were generated using a home-built noncollinear optical parametric amplifier pumped by the Ti:sapphire laser. The spectra of the various excitation pulses are compared with the UV−visible spectra of HOCbl and H2OCbl+ in Supporting Information Figure S1. Broad-band probe pulses were generated by focusing either the fundamental or second harmonic of the laser into a 3 mm translating CaF2 window. The resulting continua together span the wavelength region from 290 to 700 nm. Measurements were obtained by overlapping the pump and probe focus in a 1 mm quartz flow cell or in a wire-guided free-flowing stream of solution with a ca. 300 μm path length. The cross-correlation of pump and probe pulses ranges from 90 to 210 fs full width at half-maximum (fwhm), depending on the nature of the sample and the excitation wavelength. The shortest instrument response function (irf) is observed for wire-guided flow and visible excitation, while longer response functions are recovered for UV excitation and/ or a quartz flow cell. In all cases, the fwhm of the irf is shorter than the ca. 300−350 fs component recovered in global analysis of the data. The relative polarization of the pump and probe pulses was set to parallel, perpendicular, or the magic angle (54.7°) using a half-wave plate in the pump beam. Analysis of the transient spectra was carried out using the freely available global analysis program Glotaran20 or a more flexible pythonbased program VarPro21 developed in our laboratory. Samples were prepared by dissolving hydroxocobalamin hydrochloride (Sigma-Aldrich) to a concentration of 1.0 mM (flow cell) or 3.6 mM (wire-guided) in the appropriate buffered solution. Phosphate buffers at 12 mM with pH 5.2, 7.4, 8.0, 8.7, and 10.3 were prepared by dissolving monobasic and dibasic potassium phosphate (Sigma-Aldrich) in deionized water. Carbonate buffer at 11 mM and pH 10.6 were prepared by dissolving potassium bicarbonate (Sigma-Aldrich) and potassium carbonate anhydrous (Fluka) in deionized water. The pHs of the buffered solutions were measured by an Orion Research digital pH/millivolt meter model 611 with a 9106BNWP probe. The pKa of hydroxocobalamin is ca. 8 (values from 7.8 to 8.1 reported).19 Assuming low concentrations and activity coefficients of γ ≈1, the fraction of H2OCbl+ at pH 5.2 is 99.8%. It is 80% at pH 7.4, 50% at pH 8.0, 17% at pH 8.7, and 0.4% at pH 10.3. Extensive data collection was performed at pH 5.2 and pH 10.3 to isolate the contributions of HOCbl (vitamin B12b) and H2OCbl+ (vitamin B12a). UV−visible spectra were collected on a Shimadzu UV-2600 spectrometer. Steady-state photolysis measurements were performed using a sample prepared by dissolving the compound and an excess of radical scavenger (sodium benzoate or sorbitol) in deoxygenated buffered water solutions. The photolysis source was a xenon arc lamp with various filter combinations to define the excitation spectrum or a mercury pen lamp with a strong emission line at 253 nm to provide deep UV excitation. Computational Details. The model structures used in these calculations were derived from high-resolution X-ray crystallographic data.22,23 The full structures were truncated and simplified in order to reduce computational cost. As applied in previous studies,12,16,24 the side chains of the corrin macrocycle were replaced by hydrogens, and the axial base was simplified to be a much smaller imidazole (Figure 1). Furthermore, the simplification of the DBI base involves removal of the phosphate ion that results in a positive charge for the models. The notations for the HOCbl and H2OCbl+ model complexes are Im[CoIII(corrin)]-OH+ and Im-[CoIII(corrin)]-H2O2+, respectively. The structures of the model complexes’ optimized
Figure 1. Hydroxocobalamin (HOCbl, vitamin B12b) or aquocobalamin (H2OCbl+, vitamin B12a). The truncated model used in calculations is also shown.
chemically distinct species. HOCbl, like vitamin B12 (CNCbl), is a neutral solid with a negative ion as the axial ligand donating electrons to the positively charged cobalt ion. H2OCbl+ is a positively charged species, requiring a counterion (e.g., hydroxocobalamin hydrochloride) with a neutral water molecule acting as the upper axial ligand to the cobalt ion. At physiological pH (ca. 7.4), a solution prepared from hydroxocobalamin hydrochloride is a mixture of HOCbl and H2OCbl+ (pKa ≈ 8).19 To elucidate the photolysis mechanism at physiological pH, it is necessary to consider both HOCbl and H2OCbl+. The concentrations of HOCbl and H2OCbl+ in the sample can be modulated using buffers to change the pH. In the present contribution, we investigate the photochemistry of HOCbl and H2OCbl+ as a function of pH, ranging from 5.2 (99+ % H2OCbl+) to 10.3 (99+% HOCbl), and as a function of excitation wavelength, ranging from 269 to 575 nm. The results are compared with TD-DFT simulations of both compounds. We previously reported that at pH 10.3, where the sample is >99% HOCbl, no measurable formation of cob(II)alamin is observed for excitation wavelengths > 350 nm, while photolysis forming cob(II)alamin with low quantum yield ( ∼350 nm, but photolysis to cob(II)alamin is observed at wavelengths < 300 nm. The quantum yield for photolysis of H2OCbl+ at 269 nm (∼1.5%) is similar to that observed for HOCbl. Time-resolved absorption measurements demonstrate that the excited state dynamics of H2OCbl+ differ substantially from those observed for HOCbl. In particular, the excited state lifetime of H2OCbl+ is significantly shorter than that for HOCbl (0.35 ± 0.15 vs 5.3 ± 0.4 ps). This is corroborated by TD-DFT simulations, which reveal a seam where the S1 surface minimum and the ground state, S0, coincide. No stable S1 surface minimum is identified for H2OCbl+. Samples at intermediate pHs are well modeled as linear combinations of HOCbl and H2OCbl+.
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METHODS Experimental Details. Femtosecond transient absorption measurements were performed using a Ti:sapphire laser system operating at a 1 kHz repetition rate. A pump wavelength of 404 nm was generated as the second harmonic of the laser system, and 269 nm was generated as the third harmonic of the laser system by summing 404 nm with 808 nm. Pump wavelengths B
DOI: 10.1021/acs.jpca.8b06103 J. Phys. Chem. A XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry A geometries for the ground state (S0) and the excited state (S1) are shown in Figure S2, and the corresponding geometric parameters are gathered in Tables S1 and S2. Following our signature computational approach to cobalamins,24−26 calculations were carried out using DFT27 and TDDFT28,29 with the GGA-type BP86 functional30,31 and the TZVPP basis set for Co, C, and N and the TZVP basis set for H.32 In these calculations, the Resolution of the Identity approach for computing the electronic coulomb interaction (RI‑J)33 was applied with the corresponding auxiliary basis sets.34 The Conductor-like Screening Model (COSMO)35 with water (COSMO/H2O) as the solvent was employed. All calculations were completed using TURBOMOLE.36−40
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RESULTS AND DISCUSSION Femtosecond to picosecond transient absorption measurements were performed as a function of pH, excitation wavelength, and polarization. The excited state lifetime of H2OCbl+ is substantially shorter than the excited state lifetime of HOCbl (Figure 2). A trend of increasing lifetime with increasing pH is Figure 3. Transient absorption data obtained following excitation of H2OCbl+ (pH 5.2) with 269 nm pulses.
Figure 2. Excited state decay and bleach recovery at 530 nm following excitation of H2OCbl+/HOCbl as a function of pH. The excitation wavelength was 404 nm for these traces.
Figure 4. Long-lived photoproduct (>50 ps) following excitation of H2OCbl+ at 404 or 269 nm. The spectra plotted here represent averages from 50 to 550 ps. For comparison, the cob(II)alamin spectrum corresponding to a quantum yield for product formation of 1.5% is also plotted.
apparent in the figure. To isolate the contributions of H2OCbl+ and HOCbl, all further measurements were performed at pH 5.2 (>99% H2OCbl+) or pH 10.3 (>99% HOCbl) Photolysis of Aquocobalamin. Photolysis of H2OCbl+ at pH 5.2 using 253 nm excitation results in formation of cob(II)alamin. (See Supporting Information Figures S3 and S4.) No photolysis was detected for excitation wavelengths > 350 nm. As observed earlier for HOCbl at pH 10.3, the onset of photolysis for H2OCbl+ falls between 350 and 290 nm, and the yield is much lower than that observed for adenosylcobalamin.12 Time-resolved absorption spectra were obtained following excitation of H2OCbl+ with 269 nm for time delays out to 550 ps. The relative polarization of the pump and probe pulses was set at the magic angle to eliminate the influence of orientation. These data are dominated by ground state recovery within a few picoseconds (Figure 3). The data were fit to a model consisting of a fast component (
