Time-Resolved Spectroscopic Studies of B12 Coenzymes - American

12180

J. Phys. Chem. B 2001, 105, 12180-12188

Time-Resolved Spectroscopic Studies of B12 Coenzymes: Influence of Solvent on the Photolysis of Adenosylcobalamin Laurie M. Yoder, Allwyn G. Cole, Larry A. Walker, II, and Roseanne J. Sension* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1055 ReceiVed: June 6, 2001; In Final Form: September 4, 2001

Femtosecond-to-nanosecond transient absorption spectroscopy is used to investigate the photolysis of coenzyme B12, 5′-deoxyadenosylcobalamin, as a function of solvent environment comparing water, ethylene glycol, and mixtures of water and ethylene glycol. Photolysis in ethylene glycol is characterized by the clean formation of a cob(II)alamin species on a time scale e 28 ps. Competition between cage escape and geminate recombination of the initial radical pair leads to a nanosecond photolysis quantum yield of ca. 8%. This is in contrast to the photolysis of adenosylcobalamin in water, where an additional intermediate state is identified, and the net quantum yield for photolysis is three times higher. The additional intermediate observed in aqueous solution may correspond to a base-off alkylcobalamin or to a cob(II)alamin-like state having an enhanced rate for ground-state recovery. The competition between cage escape and geminate recombination for adenosyl and cob(II)alamin radical pairs is investigated by using mixtures of ethylene glycol and water to vary the viscosity systematically, and thereby influence the rate for escape from the initial solvent cage. The intrinsic rate constant for geminate recombination is found to be kR ) 1.39 ( 0.06 ns-1, independent of the solvent system. The effective recombination rate is solvent dependent, reflecting competition between recombination (kR), solvent-dependent cage escape (kE ) 0.46 ( 0.07 cp ns-1/η, where η is the solvent viscosity), and the formation of a caged radical pair species incapable of direct recombination (kIA ) 0.13 ( 0.06 ns-1). The most likely explanation for the inactive caged radical pair is the interconversion between singlet and triplet geminate radical pairs.

Introduction One of the key steps in the catalytic mechanism of coenzyme B12-dependent mutase enzymes is the homolytic cleavage of the carbon-cobalt bond in 5′-deoxyadenosylcobalamin to form cob(II)alamin and an adenosyl radical (Ado•). An important open question in B12 catalysis addresses the nature of the proteincoenzyme interaction and how the protein weakens the carboncobalt bond to allow facile bond cleavage. Although these enzymes function through the thermal cleavage of the carboncobalt bond, the photolysis of adenosylcobalamin has sometimes been used as a model or experimental substitute for thermal homolysis. In recent work we have used ultrafast spectroscopic techniques to explore the mechanism for photolysis of both adenosylcobalamin and the related B12 coenzyme, methylcobalamin, in an attempt to establish or refute the validity of photolysis as a model for thermolysis in studies of B12 model compounds and B12-dependent enzymes.1-4 A firm connection between thermolysis and photolysis mechanisms in alkylcobalamins, if it exists, will lay the groundwork for dynamic studies of protein-cofactor interactions, effective photoinitiated studies of enzyme mechanism in B12-dependent enzymes, and timeresolved studies of protein structure. It has long been known that photolysis of alkylcobalamins in aqueous solution under anaerobic conditions results in the homolytic cleavage of the carbon-cobalt bond to form an alkyl radical and a cob(II)alamin radical.5-7 Chen and Chance used nanosecond transient absorption spectroscopy to establish the formation of cob(II)alamin on a sub 10 ns time scale following * Corresponding author. E-mail: [email protected].

excitation of adenosylcobalamin in aqueous solution at 532 or 355 nm, and to determine a quantum yield of φ ) 0.23 ( 0.04 for the formation of cob(II)alamin.8 In a later study, this same group used continuous wave measurements with excitation at 442 nm to determine a photolysis quantum yield of φ ) 0.20 ( 0.03 for formation of cob(II)alamin from adenosylcobalamin.9 More recently, our ultrafast measurements have demonstrated that the photoproduct spectrum observed for all time delays greater than ca. 150 ps following excitation of adenosylcobalamin in aqueous solution is well-defined and corresponds to the formation of a cob(II)alamin species, suggesting that bond homolysis occurs on a picosecond time scale.2,3 These measurements have also demonstrated that the photolysis quantum yield in adenosylcobalamin is determined primarily, but not entirely, by competition between geminate recombination and diffusion to form solvent-separated radical pairs. Most of the photolysis studies of alkylcobalamins have been performed in aqueous or buffered aqueous solvents, although a few pertinent photolysis studies have been performed in alternative solvents.9-12 Of particular note, Grissom and coworkers have reported magnetic field effects on the photolysis of alkylcobalamins as a function of solvent viscosity.10,11 On the other hand, most of the thermal bond cleavage studies of alkylcobalamins and alkylcobinamides have been performed in ethylene glycol solutions.13-19 There are several reasons for this choice of solvent for thermolysis and chemical precedent studies;13 ethylene glycol is a more viscous solvent with a higher boiling point, ethylene glycol is a substrate for B12-catalyzed reactions, permitting the study of chemical precedent for secondary enzyme reactions, and the static dielectric constant

10.1021/jp012157z CCC: $20.00 © 2001 American Chemical Society Published on Web 11/02/2001

Spectroscopic Studies of B12 Coenzymes of ethylene glycol is about half that of liquid water (37 vs 78.5 at 25 °C),20 providing a more protein-like environment. Because the macroscopic dielectric properties of the solvent and the microscopic hydrogen bonding interactions of ethylene glycol versus water may influence the photolysis dynamics, it is useful to explore the influence of solvent on primary photolysis of alkylcobalamins. In this paper we use femtosecond-to-nanosecond transient absorption spectroscopy to explore the effect of the solvent environment on the photolysis of adenosylcobalamin. As a part of this study the detailed photolysis mechanisms in water and ethylene glycol are compared. These comparisons provide additional insights into the photolysis mechanism in both solvents. In addition, the competition between cage escape and geminate recombination is investigated using mixtures of ethylene glycol and water to vary the viscosity systematically, and to influence the rate for escape from the initial solvent cage. Experimental Methods Transient Absorption Measurements. Transient absorption measurements were performed using a femtosecond laser system and experimental method as described previously, with a few important modifications.1,3 Briefly, a self-mode-locked titanium sapphire oscillator, running at 100 MHz and producing 20 fs, 2 nJ pulses, was regeneratively amplified at a 1 kHz repetition rate. The resulting laser beam was centered at approximately 800 nm, providing 400 µJ, 70 fs pulses at a repetition rate of 1 kHz. Tunable probe pulses were generated by sending the 800 nm laser pulse into a home-built optical parametric amplifier constructed according to the design of Riedle and co-workers.21 For the present set of measurements the output consisted of 100to-200 fs pulses centered at peak wavelengths between 470 and 633 nm inclusive. At each wavelength a 10 nm band-pass interference filter was used to limit the spectral bandwidth of the probe pulse. For kinetic measurements the probe pulses were delayed with respect to the pump pulses by a 1.5 m computercontrolled motorized translation stage (Newport-Klinger). This stage allows measurements to be made with femtosecond resolution (1 µm step size ) 6.667 fs of delay) out to a maximum time delay of ca. 10 ns. Traces were collected from -10 ps to 9 ns using variable time steps, typically 0.1 ps (-10 to -1 ps), 0.025 ps (-1 to 1 ps), 0.050 ps (1 to 5 ps), 0.1 ps (5 to 10 ps), 0.2 ps (10 to 20 ps), 0.5 ps (20 to 50 ps), 1 ps (50 to 100 ps), 2 ps (100 to 200 ps), 5 ps (200 to 500 ps), 10 ps (500 ps to 1 ns), 20 ps (1 to 2 ns), 50 ps (2 to 9 ns). Broadband spectral measurements at selected time delays were made by using a white light continuum probe and a spectrometer for analysis as described previously.1 Sample Preparation. Adenosylcobalamin was obtained from Sigma and used without further purification. All samples were prepared and maintained under anaerobic conditions. The ethylene glycol solvent was deoxygenated by freeze-pumpthaw techniques and maintained under nitrogen until use. After addition of adenosylcobalamin and during measurements a positive-pressure argon atmosphere was used to prevent contamination with oxygen. Water used for the mixed solvents was deoxygenated by bubbling argon through doubly distilled, deionized water for a minimum of 1 h. Samples were prepared with adenosylcobalamin concentrations of ca. 2 mM. The solutions were flowed through a 1 mm path-length cell with a rate sufficient to refresh the illuminated volume between laser pulses. UV-visible spectra of the samples obtained before and after laser exposure were identical, indicating minimal photoproduct accumulation during the course of an experiment.

J. Phys. Chem. B, Vol. 105, No. 48, 2001 12181

Figure 1. Absorption spectra of adenosylcobalamin and cob(II)alamin in water (dashed lines) and ethylene glycol (solid lines) and the steadystate difference spectra obtained from the photolysis of adenosylcobalamin in water and ethylene glycol.

Steady-State Spectral Measurements. A cob(II)alamin spectrum in ethylene glycol was generated by cw photolysis from adenosylcobalamin. A 40 µM solution of adenosylcobalamin was prepared in deoxygenated ethylene glycol, in the presence of 1 mM TEMPO as a radical scavenger. The sample was handled in the dark at all times prior to measurement and an oxygen-free environment was maintained. The cob(II)alamin species was produced by illuminating the sample for 2 min intervals until no further change in the absorbance spectrum was observed. Generation of cob(II)alamin in this manner permits construction of an absolute difference spectrum for comparison with time-resolved measurements. The absorption spectrum and steady-state difference spectra comparing adenosylcobalamin and cob(II)alamin in water and ethylene glycol are shown in Figure 1. A steady-state base-off minus base-on adenosylcobalamin difference spectrum was obtained by careful preparation of adenosylcobalamin solutions of nearly identical concentration. A stock solution of 80 µM adenosylcobalamin in deoxygenated water was initially prepared. Solutions of differing pH were prepared by diluting a 5 mL aliquot of the stock solution with 5 mL of 0.02 M HCl or 5 mL of doubly distilled deoxygenated water. To ensure that the relative intensities are correct, the measurement was repeated 5 times on a freshly prepared sample each time. The pH of the base-off adenosylcobalamin was 2.20 and that of the base-on adenosylcobalamin was 5.25. All samples were handled in the dark and used immediately after preparation. Results Adenosylcobalamin in Ethylene Glycol. Transient absorption traces were obtained following excitation of adenosylcobalamin in ethylene glycol at 400 nm using probe wavelengths centered at 470, 500, 520, 540, 550, 560, 570, 600, and 633 nm. In addition, transient difference spectra between 480 and 640 nm were obtained at time delays of 40 and 900 ps. Typical transient absorption traces are shown in Figure 2. The steadystate and transient difference spectra were used to scale the transient absorption traces obtained at specific wavelengths. In this manner, the overall evolution of the transient difference spectrum may be reconstructed from the narrow bandwidth traces as illustrated in Figure 3. The evolution of the difference spectrum in ethylene glycol is qualitatively similar to the evolution of the spectrum in water.2 There is a sub 100 ps growth

12182 J. Phys. Chem. B, Vol. 105, No. 48, 2001

Yoder et al. TABLE 1: Rate Constants Determined from Analysis of the Transient Absorption Data solvent

ethylene glycol

watera

k1 or kB k2 or kI k3 k4 φ kR kE kR′ φnet

0.70 ( 0.05 ps-1 0.036 ( 0.001 ps-1 9.8 ( 0.6 ns-1 1.45 ( 0.13 ns-1 0.077 ( 0.020 1.34 ( 0.13 ns-1 0.11 ( 0.03 ns-1

0.69 ( 0.05 ps-1 0.071 ( 0.001 ps-1 9.0 ( 1.0 ns-1 2.0 ( 0.2 ns-1 0.284 ( 0.020 1.43 ( 0.21 ns-1 0.57 ( 0.06 ns-1 3.4 ( 0.3 ns-1 0.18 ( 0.02

a Rate constants k1, k2, k3, and k4 and the quantum yield φ reported for water are from ref 3.

Figure 2. Typical transient absorption traces obtained for adenosylcobalamin in ethylene glycol at the indicated probe wavelengths.

Figure 3. Solid lines: Transient difference spectra obtained at 40 and 900 ps following excitation of adenosylcobalamin in ethylene glycol at 400 nm. Symbols: Evolution of the transient difference spectrum from 500 fs to 9 ns. In the left-hand panel data are plotted for 500 fs (O), 2 ps (b), 5 ps (4), 10 ps (2), 20 ps (3), 30 ps (1), 50 ps (]) and 100 ps ([). In the right-hand panel data are plotted for 100 ps ([), 200 ps (0), 300 ps (9), 400 ps (+), 500 ps (+), 700 ps (O), 900 ps (b), 1.2 ns (4), 1.6 ns (2), 2.5 ns (3) and 9 ns (1).

of the ground-state bleach followed by a sub-nanosecond recovery to long-lived plateau. To develop a more quantitative picture of the spectral evolution, the data were fitted by using a global analysis algorithm to a model consisting of an instrument-limited Gaussian spike, four exponential decay components with rate constants ki and amplitudes Si(λ), and a nondecaying component with amplitude SP(λ). The rate constants (time constants in parentheses) obtained for the four exponential components are the following: 0.70 ( 0.05 ps-1 (1.4 ps), 0.036 ( 0.001 ps-1 (28 ps), 9.8 ( 0.6 ns-1 (102 ps), 1.45 ( 0.13 ns-1 (690 ps). Errors indicated for these rate constants are estimated from the results of the global analysis. The values obtained in ethylene glycol are compared with those obtained in water in Table 1. To illustrate the quality of the fit over the entire time window, a transient absorption trace obtained with a 540 nm probe is plotted on a logarithmic time scale in Figure 4 along with the calculated fit and the residuals. Decay-associated difference spectra defined by the amplitudes, Si(λ), obtained from the global fits of the data in ethylene glycol and in water are plotted in Figure 5. Error estimates for the decay-associated spectra illustrated in this figure are determined from the reproducibility of the amplitudes in the

Figure 4. Transient absorption trace obtained at 540 nm following excitation of adenosylcobalamin in ethylene glycol at 400 nm. The early behavior is plotted on a linear time scale in the left-hand panel. The evolution from 500 fs to 9 ns is plotted on a logarithmic time scale in the right-hand panel. The fit (black dashed line) and residuals are also shown in each panel. The horizontal solid black line marks ∆A ) 0.

Figure 5. Decay-associated difference spectra obtained from the global analysis of the transient absorption traces in ethylene glycol (left-hand panel) and water (right-hand panel). The symbols represent: -+- k1, -b- k2, -3- k3, -[- k4, -0- ∞ (“permanent” offset). Estimated error bars obtained from the global analysis of the transient absorption data are shown for the 470 nm k4 points. The absolute magnitude of the estimated error is constant for all of the points.

global analysis and from the reproducibility evident between different traces obtained with the same probe wavelength. The dominant decay component, S4(λ), observed in both solvents following excitation of adenosylcobalamin corresponds to the geminate recombination of cob(II)alamin and adenosyl radical. The relative amplitude of this geminate recombination component to the long-lived plateau, SP(λ), is essentially independent

Spectroscopic Studies of B12 Coenzymes

Figure 6. Transient absorption traces obtained at 540 nm following excitation of adenosylcobalamin in ethylene glycol/water mixtures at 400 nm. The early behavior is plotted on a linear time scale in the left-hand panel. The evolution from 500 fs to 9 ns is plotted on a logarithmic time scale in the right-hand panel. The fit (black dashed lines) and residuals are also shown in each panel. The horizontal solid black line marks ∆A ) 0 for each trace. The premixing volume percent ethylene glycol is indicated with each plot.

of probe wavelength. In addition, the rate constant is well determined at each wavelength and essentially independent of probe wavelength even when the data sets are fit independently. These results demonstrate that cage recombination occurs on a time scale of 690 ( 60 ps in ethylene glycol, approximately 40% slower than for adenosylcobalamin in water (500 ( 50 ps). Adenosylcobalamin in Mixtures of Water and Ethylene Glycol. Transient absorption traces were obtained following excitation of adenosylcobalamin in mixtures of ethylene glycol and water at 400 nm using probe wavelengths centered at 520, 540, 550, and 560 nm. In both water and ethylene glycol the dynamics observed at these wavelengths, near the maximum of the ground-state bleaching, are dominated by the geminate recombination of radical pairs. Thus, these wavelengths provide a data set sufficient to explore the effect of solvent viscosity on geminate recombination. Samples used in these studies were prepared as 3:1, 1:1, and 1:3 mixtures of ethylene glycol and water by volume prior to mixture. Typical data obtained at 540 nm are plotted on a logarithmic scale in Figure 6 to illustrate the temporal behavior over the entire time window. The data for each solvent mixture were fitted by using a global analysis algorithm to a model consisting of an instrument-limited Gaussian spike, four exponential decay components, and a nondecaying component as described above for pure ethylene glycol solvent. The fits and residuals at 540 nm are plotted in Figure 6. Although the absolute time constants vary somewhat, all of the ethylene glycol/water mixture data sets require a time constant between 1.5 and 3.5 ps, a time constant around 30 ( 10 ps, a time constant between 100 and 200 ps, and a time constant between 600 and 700 ps. For ease of comparison, estimated decay-associated spectra over the spectral region between 520 and 560 nm are plotted in Figure 7 for the five solutions studied here (water solvent, ethylene glycol solvent, and the three solvent mixtures). Because the available data for the mixtures are insufficient to determine accurate decay-associated spectra, these estimated spectra were obtained by normalizing the dominant 500 to 700 ps component to the average of the relative intensities observed in ethylene glycol and water (see Figure 5). This allows qualitative comparison of the similarities and differences between the solvent systems.


Figure 7. Qualitative decay-associated difference spectra between 520 and 560 nm for each of the five solvent mixtures investigated here. The symbols represent: -+- k1, -b- k2, -3- k3, -[- k4, -0- ∞ (“permanent” offset). The premixing volume percent ethylene glycol in water is indicated in each panel. For ease of comparison across solvent mixtures the data have been scaled such that the k4 amplitudes are -1.00 at 520 nm, -0.975 at 540 nm, -0.847 at 550 nm, and -0.702 at 560 nm. These values represent averages of the observed relative amplitudes in ethylene glycol and water. The solid horizontal line in each panel represents the zero amplitude level (no signal).

Figure 8. Model for the photolysis of adenosylcobalamin as described in the text.

In pure water and all of the water/ethylene glycol mixtures the behavior is qualitatively the same. On the other hand, the ca. 28 and 100 ps components are significantly different in pure ethylene glycol. The relative amplitude of the ca. 28 ps component (filled circles) is much smaller in ethylene glycol than in any of the other solvents. More importantly, the ca. 100 ps component (open triangles) corresponds to the decay of an absorption or growth of a bleach in ethylene glycol, but to the growth of an absorption or decay of a bleach in the other four solvent systems. This is a significant difference, discussed in greater detail below. Discussion Photolysis Mechanism in Ethylene Glycol. The model used in our earlier papers to account for the evolution of adenosylcobalamin in water is illustrated in Figure 8.2,3 Following excitation, one or more excited states are populated. These excited states decay sequentially to produce an intermediate state designated I on a time scale of 14 ps in water and 30 ( 10 ps in ethylene glycol and in the three ethylene glycol/water mixtures studied here. The excited states are designated {AdoCob}*, {A} and {B} in Figure 8. These states represent a


Yoder et al.

minimal set required to account for the observed data. The initial excited state produced by photoexcitation ({Ado-Cob}*) decays on a ca. 100 fs time scale to form the state designated {A}. This initial decay component is not fully resolved in the data presented here, but is contained in the instrument-limited spike. Data obtained for aqueous adenosylcobalamin with a 40 fs instrument function (ca. 28 fs pulses) exhibit a clear 92 ( 8 fs decay component.4 The intermediate excited state designated {A} decays on a ca. 1-3 ps time scale to form {B} which in turn decays on a 14-37 ps time scale to produce the intermediate state {I}. It should be noted that the states {A} and {B} may represent distinct intermediate electronic states or may evince the thermal and conformational relaxation of adenosylcobalamin following excitation. As discussed in our earlier papers, the intermediate state {I} has a spectrum that is consistent with a cob(II)alamin species.2,3 In aqueous solution, this intermediate decays with a rate constant, kR′ + kH, to recombine or to form a relaxed radical pair Ado‚ + Cob(II)alamin. Competition between cage escape and geminate recombination of the relaxed radical pair accounts for the ultimate photolysis quantum yield for solvent-separated radical pairs. Although one may imagine more complicated models to account for the observed data, the model outlined in Figure 8 appears to be most straightforward. Given a model for the dynamics following excitation of adenosylcobalamin it is possible to use the measured rate constants (ki) and the observed decay-associated spectra (Si(λ) in Figure 5) to construct the species-associated difference spectra and the species-associated spectra for each of the intermediate states. For the model in Figure 8, assuming that kA is much larger than all of the succeeding rate constants, the speciesassociated difference spectra are given by

δ{A}(λ) ) S1(λ) + S2(λ) + S3(λ) + S4(λ) + SP(λ) (1)

( )

( ) ( )

(

)

k1 - k2 k1 - k3 δ{B}(λ) ) S2(λ) + S3(λ) + k1 k1 k1 - k4 S4(λ) + SP(λ) (2) k1 and

δ{I}(λ) )

(k2 - k3)(k1 - k3) S3(λ) + k1k2 (k2 - k4)(k1 - k4) S4(λ) + SP(λ) (3) k1k2

(

)

for the various intermediate states observed following excitation. The spectrum of the caged, {RP}, and solvent-separated radical pair, RP, states are given by

δ{RP}(λ) )

k3 kH

[(

)

(k3 - k4)(k2 - k4)(k1 - k4) S4(λ) + k1k2k3

]

SP(λ) (4) and

δRP(λ) )

k3 k4 S (λ) kH kE P

(5)

In eqs 1-5, the rate constants obtained in the global analysis are equated to the rate constants in the model as k1 ) kB, k2 )

Figure 9. Left hand panel: Species-associated difference spectra obtained for adenosylcobalamin in ethylene glycol as described in the text, -O- {A}, -0- {B}, -[- {I}, -1- {RP}. The red solid line is the steady-state difference spectrum in ethylene glycol. For clarity the spectrum of the solvent-separated radical pair, RP, is not shown. This spectrum is noisy due to the inherent low amplitude of SP(λ), but is essentially the same as the spectrum of {RP}. Right-hand panel: Species-associated spectra obtained by adding back the component due to the bleaching of ground-state adenosylcobalamin. The red solid lines are the steady-state spectra of cob(II)alamin and adenosylcobalamin in ethylene glycol. Error bars (green) for the {A} and {RP} spectra are indicated at 470 nm in each panel. These were estimated by propagation of errors through eqs 1-4. Errors for the {B} and {I} spectra are intermediate between the extremes illustrated.

kI, k3 ) kH + kR′ and k4 ) kR + kE. The spectra of the caged radical pair and the solvent-separated radical pair are identical. Thus, eqs 4 and 5 can be used to determine the quantum yield, φ ) kE/k4, for cage escape by relaxed radical pairs.

φ)

QR 1 + QR

(6)

where R ) SP(λ)/S4(λ) and Q ) k1k2k3/[(k1 - k4)(k2 k4)(k3 - k4)]. Species-associated spectra for the photolysis of adenosylcobalamin in ethylene glycol are shown in Figure 9. The assumptions in this calculation are: (1) the model in Figure 8 accurately describes the dynamics; (2) kR′ , kH implying that k3 ≈ kH; and (3) the spectra of the caged radical pair and the solvent-separated radical pair are identical. Under these assumptions there are no open parameters in deriving the speciesassociated spectra. All of the parameters are uniquely determined by the global analysis of the transient absorption data. The assumptions described above are supported by the results. The spectra of {I}, {RP}, and RP (not shown in Figure 9) are essentially identical to each other and to the steady-state spectrum of cob(II)alamin. The transition from {I} to {RP} is characterized primarily by a small growth in the intensity of the cob(II)alamin peak absorption around 470 nm. The spectra of the intermediates {A} and {B} exhibit an evolution from an adenosylcobalamin or cob(III)alamin type spectrum5,6 to a cob(II)alamin type spectrum. It is clear from the species-associated spectra in Figure 9, that photoinduced bond homolysis of adenosylcobalamin in ethylene glycol is complete on a time scale e 28 ps. The states {I}, {RP}, and RP are clearly characterized by the presence of cob(II)alamin. The slight evolution from {I} to {RP} is consistent with a modest relaxation of the corrin ring following bond cleavage. The states {A} and {B} are not as clearly identified. These states may represent distinct intermediate electronic states. On the other hand, the spectrum of


Figure 10. Left-hand panel: Species-associated difference spectra obtained for adenosylcobalamin in water as described in the text, -O{A}, -0- {B}, -[- {I}, -1- {RP}. The red solid line is the steady-state difference spectrum in water. For clarity the spectrum of the solventseparated radical pair, RP, is not shown. This spectrum is essentially the same as the spectrum of {RP}. Right-hand panel: Species-associated spectra obtained by adding back the component due to the bleaching of ground-state adenosylcobalamin. The red solid lines are the steadystate spectra of cob(II)alamin and adenosylcobalamin in water. Error bars (green) for the {A} and {RP} spectra are indicated at 470 nm in each panel. These were estimated by propagation of errors through eqs 1-4. Errors for the {B} and {I} spectra are intermediate between the extremes illustrated.

{B} suggests that this state may evince rapid relaxation of the initially formed cob(II)alamin. A more complete transient absorption study extending into the near-ultraviolet, or a different type of measurement, directly sensitive to the bond cleavage, will be required to assign the states {A} and {B} conclusively. Comparison of Photolysis Mechanism in Ethylene Glycol and Water. The decay-associated difference spectra obtained in water and ethylene glycol are compared in Figure 5. As mentioned above, there are two significant differences between the decay-associated difference spectra in these two solvents. First, the relative amplitude of the 14 ps component in water is substantially larger than the relative amplitude of the 28 ps component in ethylene glycol. Second, but more importantly, the ca. 100 ps component has a different spectral shape, at many wavelengths a different sign, and a much larger amplitude in water than in ethylene glycol. These differences have a significant effect on the calculated species-associated spectrum for the state {I} according to the model in Figure 8. In our earlier papers, the large negative amplitude observed for S3(λ) was attributed to partial ground-state recovery from the state {I} (i.e., kR′ not negligible).2,3 However, a value for kR′ comparable to kH is not required to explain the data obtained in ethylene glycol solution. In light of these differences, it is useful to reexamine the species-associated spectra in aqueous solution and consider the possibility of alternative explanations for the observed spectral evolution. The data obtained in ethylene glycol and in water can be best compared by performing equivalent analyses on the two data sets. Species-associated spectra in water calculated under the same assumptions described above for adenosylcobalamin in ethylene glycol are plotted in Figure 10. In contrast to the results described for ethylene glycol, the spectrum of {I} in water exhibits a significant increase in the bleach of the adenosylcobalamin band over that expected for the formation of cob(II)alamin. At least two different hypotheses may explain these observations.


Figure 11. Spectra of base-off adenosylcobalamin (pH 2.5) and baseon adenosylcobalamin (pH 5.25) and the estimated difference spectrum for the formation of base-off adenosylcobalamin from base-on adenosylcobalamin. The dashed lines are the steady-state spectra of adenosylcobalamin, cob(II)alamin, and the steady-state difference spectrum for the formation of cob(II)alamin from adenosylcobalamin at neutral pH. The filled diamonds represent the species-associated difference spectrum and the open diamonds represent the species-associated spectrum for the intermediate species {I} observed in the photolysis of adenosylcobalamin in water.

The first possibility is that the intermediate species {I} is in fact different in water than in ethylene glycol and that the spectrum of {I} in water is blue-shifted with respect to the cob(II)alamin spectrum as illustrated in the right-hand panel of Figure 10. In this context it is interesting to note that such a blue shift may be observed, for example, if the intermediate species arises from the dissociation of the nitrogenous axial ligand.22 Thus, one hypothesis capable of accounting for the observed data is that in aqueous solvent the intermediate {I} corresponds to base-off adenosylcobalamin and the conversion from {I} to {RP} represents reattachment of the nitrogen ligand and homolysis of the carbon-cobalt bond. The plausibility of this hypothesis may be tested by comparing an estimated base-off minus base on difference spectrum with the species-associated difference spectrum for {I}. The steadystate base-off spectrum may be generated in the laboratory by lowering the pH of adenosylcobalamin in water.22 The calculated transient species-associated spectra are compared in Figure 11 with those predicted by using the spectrum of the base-off species obtained at low pH. The observed spectral shift is somewhat larger than that predicted from the low pH spectrum. In addition, intensity is observed at long wavelengths in the spectrum of {I}, that is not observed in the low pH spectrum. However, the overall agreement is quite good. Clearly the hypothesis that the intermediate species is base-off adenosylcobalamin must be considered plausible. An alternative hypothesis, also capable of accounting for the observed data, is to release the assumption that kR′ , kH and use the ratio k3/kH in eqs 4 and 5 as a fitting parameter to bring the spectrum of {I} into line with the equivalent spectrum observed in ethylene glycol. This hypothesis represents the model favored in our earlier papers.2,3 The species-associated spectra derived under this assumption are shown in Figure 12, with k3/kH ) 1.6, kR′ ) 3.4 ns-1, and kH ) 5.6 ns-1. Under this hypothesis the quantum yield for the formation of the relaxed radical pair {RP} is ca. 0.62 and the net quantum yield for photodissociation is 0.18. The spectra and photolysis yields deduced under this second hypothesis are reasonable and this hypothesis must also be considered plausible.


Figure 12. Left-hand panel: Species-associated difference spectra obtained for adenosylcobalamin in water as described in the text with k3/kH as a fitting parameter, -O- {A}, -0- {B}, -[- {I}, -1- {RP}. The red solid line is the steady-state difference spectrum in water. Righthand panel: Species-associated spectra obtained by adding back the component due to the bleaching of ground-state adenosylcobalamin. The red solid lines are the steady-state spectra of cob(II)alamin and adenosylcobalamin in water. Error bars (green) for the {A} and {RP} spectra are indicated at 470 nm in each panel. These were estimated by propagation of errors through eqs 1-4. Errors for the {B} and {I} spectra are intermediate between the extremes illustrated.

Viscosity Dependence of Cage Escape and Geminate Recombination. The spectral range over which data were obtained in the mixtures of ethylene glycol and water is not sufficient to determine accurate species-associated spectra for each solvent mixture. It is apparent from the decay-associated spectra shown in Figure 7, however, that the photolysis mechanism in the mixtures more nearly resembles that observed in water than that observed in ethylene glycol. The amplitude of S3(λ) is large and uniformly negative between 520 and 560 nm. This observation suggests that the presence of water interacting with the adenosylcobalamin influences the photolysis mechanism. The presence of water to solvate the 5′-deoxyadenosyl group and/or the dimethylbenzimidazole axial ligand may activate a channel to facilitate the formation of a transient base-off intermediate species as required by the first hypothesis in the preceding section, or facilitate recombination and groundstate recovery from the intermediate state {I} as in the second hypothesis. In either of these two mechanisms for photolysis, the k4, 500 ps to 690 ps, decay component corresponds to geminate recombination of relaxed adenosyl and cob(II)alamin radical pairs. If cage escape by the adenosyl radical is diffusion limited, the rate constant for cage escape, kE in Figure 8, should scale linearly with the inverse of the solvent viscosity. The quantum yield, φ, for cage escape by relaxed radical pairs may be obtained from the amplitudes and rate constants according to eq 6. The rate constants for cage escape and geminate recombination may be calculated from kE ) k4φ and kR ) k4 kE. These values are summarized in Table 2 for all of the solvent systems investigated here. The intrinsic rate constant for geminate recombination of adenosyl radical and cob(II)alamin is essentially independent of the solvent with an average value of 1.39 ( 0.06 ns-1. The rate constant for cage escape exhibits a clear dependence on the solvent environment. Values for the viscosity of mixtures of ethylene glycol and water as a function of percent ethylene glycol by weight are tabulated in ref 20. The values reported in this table, with interpolation when necessary, were used to estimate the viscosity for each of the solvent mixtures studied here. These estimated

Yoder et al. viscosities are summarized in Table 2. The observed rate constants are plotted versus 1/η in Figure 13. The dependence of the rate constant for cage escape on inverse viscosity is well described by a line with a slope of 0.46 ( 0.07 ns-1 cp and an intercept of 0.13 ( 0.04 ns-1. The errors in these quantities are estimated using propagation of errors in linear regression assuming that the error in the rate constant kE is 0.06 for all samples. In the simplest picture, the effective rate constant for recombination, k4, should be the sum of an intrinsic rate constant for recombination and a viscosity-dependent rate constant for cage escape approaching zero at infinite viscosity. The finite intercept, 0.13 ( 0.04 ns-1, implied by the experimental data suggests that this model is overly simplified. Intrinsic in the simple model are assumptions about the orientation dependence (or lack thereof) for the geminate recombination of adenosyl radical with cob(II)alamin, the time scale for reorientation of the adenosyl radical within the cage, and the time scale for evolution from a singlet to a triplet radical pair. The simple model will hold if there is no orientation dependence to the recombination or the reorientation of adenosyl radical within the cage is much faster than kR-1 or much slower than kE-1 and if the time scale for evolution of the radical pair between the singlet and triplet states is much faster than kR-1 or much slower than kE-1. Of course, the adenosyl radical has a preferred orientation for recombination with cob(II)alamin and one would anticipate reorientation of the adenosyl radical within the solvent cage on a finite time scale. In addition, evolution between singlet and triplet radical pair states may reasonably be expected on a nanosecond time scale. In fact, magnetic-field-dependent photolysis quantum yields and recombination rates reported by Grissom and co-workers demonstrate that singlet-triplet interconversion competes with radical pair recombination.10,11 In the investigations of Grissom and co-workers, both picosecond time-resolved absorption measurements and cw photolysis measurements were used to probe magnetic field effects on radical recombination in alkylcobalamins. A magneticfield-dependent effective recombination rate constant is reported for both adenosylcobalamin in water and adenosylcobalamin in 75% glycerol (η ≈ 30).10 However, the relative amplitudes of the decaying and long-lived plateau components are not reported, and the data do not allow resolution of the multiexponential decay reported here, making interpretation of the apparent rate changes in these picosecond measurements problematic. In addition, the viscosity-independent zero-field rate constant for geminate recombination (1.1 ( 0.06 ns-1), reported by Grissom and co-workers, is in conflict with the clear viscosity dependence observed in the present work. A more detailed study of the proposed magnetic field effect on the effective rate for geminate recombination is necessary to interpret the apparent field-dependent change in rate constant. In contrast to the apparent results of the picosecond measurements, the cw quantum yield for the photolysis of adenosylcobalamin in water at 514 nm is unaffected by a magnetic field of up to 0.2 T while the photolysis quantum yield decreases by ca. 30% in 75% glycerol.10 In a second paper by Grissom and co-workers, the rate for cw photolysis in 80% glycerol decreases by ca. 20% in 0.05 T-0.6 T fields.11 On the basis of the results of their picosecond and cw measurements, Grissom and co-workers suggest that a magneticfield-dependent recombination probability for the initial solventseparated radical pair is the primary factor responsible for the observed dependence of quantum yield on magnetic field.10,11,23



TABLE 2: Influence of Viscosity on Cage Escape and the Quantum Yield for Geminate Recombination in Water/Ethylene Glycol Mixtures % ethylene glycola

viscosityb (cp)

k4 (ns-1)

φ (at 9 ns)

kR (ns-1)

kE (ns-1)

100% 75% 50% 25% 0%

19.9 9 4.1 2.0 1.0

1.45 ( 0.13 1.62 ( 0.04 1.70 ( 0.1 1.74 ( 0.08 2.0 ( 0.2

0.077 ( 0.020 0.127 ( 0.020 0.15 ( 0.03 0.24 ( 0.03 0.284 ( 0.020

1.34 ( 0.13 1.41 ( 0.05 1.45 ( 0.11 1.33 ( 0.10 1.43 ( 0.21

0.11 ( 0.03 0.205 ( 0.030 0.25 ( 0.05 0.41 ( 0.06 0.57 ( 0.06

a Percent ethylene glycol by volume prior to mixture. b Relative viscosity at 20 °C is tabulated in ref 20 for mixtures of water and ethylene glycol as a function of percent ethylene glycol by weight. The viscosities reported here are interpolated from the table in ref 20.

Figure 13. The rate constants for geminate recombination and cage escape of relaxed radical pairs formed following excitation of adenosylcobalamin at 400 nm are plotted as a function of inverse viscosity (1/η).

Figure 14. Schematic model for the geminate recombination and cage escape of adenosyl radicals taking into account singlet-triplet conversion of the radical pair. kE as reported in Tables 1 and 2 and Figure 13 will contain contributions from pure diffusive cage escape (kD) and singlet-triplet interconversion. A similar model could be proposed if the inactive caged radical pair is attributed to reorientation or relaxation rather than intersystem crossing.

In this interpretation, the effect of magnetic field on the several hundred picosecond geminate recombination of radical pairs is mitigated by slow escape from the solvent cage. However, this interpretation is not consistent with the viscosity dependence observed for k4 or φ (9 ns) in the transient absorption measurements presented here. The effect of interconversion between singlet and triplet radical pairs on the viscosity-dependent geminate recombination of adenosylcobalamin may be approximated as illustrated in Figure 14. On the

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