Ultrafast excited-state dynamics and photolysis in base-off B12 coenzymes and analogues: Absence of the trans-nitrogenous ligand opens a channel for rapid nonradiative decay

Article abstract of DOI:10.1021/jp104641u

Ultrafast transient absorption spectroscopy was used to investigate the photochemistry of adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and n-propylcobalamin (PrCbl) at pH 2 where the axial nitrogenous ligand is replaced by a water molecule. The evolution of the difference spectrum reveals the internal conversion process and spectral characteristics of the S₁ excited state. The photolysis yield in the base-off cobalamins is controlled by competition between internal conversion and bond homolysis. This is in direct contrast to the process in most base-on alkylcobalamins where primary photolysis occurs with near unit quantum yield and the photolysis yield is controlled by competition between diffusive separation of the radical pair and geminate recombination. The absence of the axial nitrogenous ligand in the base-off cobalamins modifies the electronic structure and opens a channel for fast nonradiative decay. This channel competes effectively with the channel for bond dissociation, dropping the quantum yield for primary radical pair formation from unity in base-on PrCbl and AdoCbl to 0.2 ± 0.1 and 0.12 ± 0.06 in base-off PrCbl and AdoCbl, respectively. The photolysis of base-off MeCbl is similar to that of base-off AdoCbl and PrCbl with competition between rapid nonradiative decay leading to ground state recovery and formation of a radical pair following bond homolysis.

Full text of DOI:10.1021/jp104641u

12398
J. Phys. Chem. B 2010, 114, 12398–12405
Ultrafast Excited-State Dynamics and Photolysis in Base-Off B12 Coenzymes and Analogues:
Absence of the trans-Nitrogenous Ligand Opens a Channel for Rapid Nonradiative Decay
Jian Peng, Kuo-Chun Tang, Kaitlin McLoughlin, Yang Yang, Danika Forgach, and
Roseanne J. Sension*
Department of Chemistry and Department of Physics, UniVersity of Michigan, Ann Arbor,
Michigan 48109-1055
ReceiVed: May 20, 2010; ReVised Manuscript ReceiVed: July 12, 2010
Ultrafast transient absorption spectroscopy was used to investigate the photochemistry of adenosylcobalamin
(AdoCbl), methylcobalamin (MeCbl), and n-propylcobalamin (PrCbl) at pH 2 where the axial nitrogenous
ligand is replaced by a water molecule. The evolution of the difference spectrum reveals the internal conversion
process and spectral characteristics of the S1 excited state. The photolysis yield in the base-off cobalamins is
controlled by competition between internal conversion and bond homolysis. This is in direct contrast to the
process in most base-on alkylcobalamins where primary photolysis occurs with near unit quantum yield and
the photolysis yield is controlled by competition between diffusive separation of the radical pair and geminate
recombination. The absence of the axial nitrogenous ligand in the base-off cobalamins modifies the electronic
structure and opens a channel for fast nonradiative decay. This channel competes effectively with the channel
for bond dissociation, dropping the quantum yield for primary radical pair formation from unity in base-on
PrCbl and AdoCbl to 0.2 ( 0.1 and 0.12 ( 0.06 in base-off PrCbl and AdoCbl, respectively. The photolysis
of base-off MeCbl is similar to that of base-off AdoCbl and PrCbl with competition between rapid nonradiative
decay leading to ground state recovery and formation of a radical pair following bond homolysis.
Introduction
Vitamin B12 (CNCbl) (Figure 1) is an essential human
nutrient, a precursor to the alkylcobalamins incorporated into
two human enzymes: methylmalonyl-CoA mutase and methion-
ine synthase.1-4 Methylcobalamin (MeCbl) functions as a methyl
cation donor in methionine synthase and other methyltransferase
enzymes.2 Bond homolysis of the carbon-cobalt bond in 5′-
deoxyadenosylcobalamin (coenzyme B12, AdoCbl) functions as
a source of organic radicals in cobalamin-dependent enzymes
catalyzing radical rearrangement reactions.1,4 Two major con-
formations of the B12 coenzymes are found in active enzymes:
a “base-on” form where the bottom axial ligand to the cobalt is
supplied by the dimethylbenzimidazole, as shown in Figure 1,
and a “base-off/His-on” conformation in which a histidine
residue in the protein provides the axial ligand. A small number
of methyltransferase proteins lack an axial nitrogenous ligand,
and recent work has demonstrated the presence of a water ligand
axial to the methyl.2,5-7
In addition to the active B12 dependent enzymes, there are
transport and transfer proteins that carry the necessary cobalamin
cofactors, moving them through the body and preparing the
cofactors for the enzymes. One such protein, human adenos-
yltransferase (ATR), binds cob(II)alamin, adds the 5'-deoxy-
Figure 1. Structure of cobalamins (RCbl). The R group is cyano in
adenosyl group, and delivers AdoCbl to methylmalonyl-CoA
mutase. Recent spectroscopic and structural studies of ATR have
demonstrated that this compound binds the cobalamin cofactor
in a base-off conformation without an axial nitrogenous ligand
in both the cob(III)alamin and cob(II)alamin oxidation states.8-10
The surprising presence of “base-off” cobalamin in ATR has
vitamin B12, methyl in methylcobalamin, and 5′-deoxyadenosyl in
adenosylcobalamin.
led to increased interest in the influence of the axial ligand on
the active C-Co bond.
The influence of the protein environment and axial ligand in
stabilizing or destabilizing the Co-C bond remains a question
for investigation. Many spectroscopic and theoretical studies
* To whom correspondence should be addressed. E-mail: rsension@
umich.edu.
10.1021/jp104641u  2010 American Chemical Society
Published on Web 09/03/2010

Base-Off B12 Coenzymes and Analogues
J. Phys. Chem. B, Vol. 114, No. 38, 2010 12399
have sought to understand the relationship between the electronic
structure, geometry, and environment of cobalamin cofactors.11
The cobalamin cofactor is a large molecule with a somewhat
flexible ring structure. Density functional theory (DFT) is the
method of choice, and time-dependent density functional theory
(TDDFT) is required to model the excited state spectrum.
Brunold and co-workers have combined TDDFT calculations
with extensive spectroscopic measurements to describe the
electronic structure of cobalamins in solution and bound to a
variety of proteins.5,10,12-16
Recent calculations by Kozlowski and co-workers have
explored the electronic structure and photochemistry of base-
on and base-off cobalamins in detail.17-21 These calculations
highlight some of the difficulties in modeling such a complex
molecule; the B3LYP functional appropriate for modeling the
π f π* transitions of the corrin ring is not appropriate for
the d/π f π*/d transitions sensitive to the axial ligation of the
cobalt. The BP86 functional does a better job of modeling
transitions relevant to the reactivity of the cobalt axial ligands.
This functional was used to interpret the distinctive photochem-
istry of MeCbl.17,20 Similar TDDFT studies of AdoCbl demon-
strate an important distinction between simple alkyl ligands and
the more complicated 5′-deoxyadenosyl ligand.18 Ligand-ring
charge transfer transitions (LRCT) involving Ado f π*
transitions are predicted to contribute to the low-lying states.
In a solvent-free model with the BP86 functional, the lowest
energy transition is a LRCT transition. These transitions shift
to higher energy when a polarizable continuum model (PCM)
is used to incorporate solvent effects but still fall within the
visible region of the spectrum.
Figure 2. Absorption spectra of adenosylcobalamin (green), propyl-
cobalamin (red), methylcobalamin (dark blue), and cob(II)alamin (light
blue). The inset shows the spectrum of cob(II)alamin obtained by
photolyzing AdoCbl at pH 7 and then adding acid (dark blue dashed
line) and by photolyzing AdoCbl at pH 2 (light blue solid line). The
shoulder around 540 nm varies depending on the method used to form
cob(II)alamin and may represent the contribution of an oxidation
product.
lengthens and suggest that this state mediates the bond dis-
sociation. Differences in the depth of the S1 minimum and
location of the 3(σCo-C f σ*Co-C) crossing influence the
observed rate for dissociation. The calculations predict that
dissociation will produce a triplet radical pair. In contrast, recent
steady state photolysis measurements investigating the magnetic
field effect on the recombination of the geminate radical pair
suggest that the majority of radical pairs produced following
excitation of both AdoCbl and MeCbl are formed in the singlet
state.25 The mechanism for photolysis in B12 coenzymes and
analogues remains a phenomenon requiring investigation, both
computational and experimental.
Photoexcitation has proven to be a useful technique in the
investigation of B12 coenzymes both in solution and protein
bound.22-27 These studies provide insight into the electronic
structure of cobalamins and provide a means to produce and
study the geminate radical pair. We have reported time-resolved
studies of the photolysis of both native AdoCbl28-31 and
MeCbl32,33 coenzymes as well as the ethylcobalamin (EtCbl)
and n-propylcobalamin (PrCbl) analogues.34,35 These studies
demonstrate that photolysis of AdoCbl is insensitive to excitation
wavelength (400 nm vs 520 nm), producing a geminate radical
pair within 100-130 ps at room temperature.28,29,31 The nature
of the S1 excited state from which the radical pair is formed
depends on the environment and exhibits different spectral
features in water, ethylene glycol, and the glutamate mutase
active site.28,29,35,36 The photolysis of MeCbl is wavelength-
dependent with both prompt dissociation and formation of a
long-lived S1 state (g1 ns) following excitation at 400 nm but
only formation of the long-lived S1 state following excitation
at 520 nm.32,33,36 The spectral features of the excited state are
the same in all environments studied: water, ethylene glycol,
and methionine synthase.32,35 Ethyl- and n-propylcobalamin form
a geminate radical pair within 50 ps following excitation at both
400 and 520 nm. A contribution arising from rapid dissociation
is observed following excitation at 400 nm, while a short-lived
S1 state (<50 ps), spectrally similar to that observed following
excitation of MeCbl, is more prominent following excitation at
520 nm.34,35
One straightforward way to expand the experimental database
of information available is to explore the photochemistry and
photophysics of alkylcobalamins in a variety of different states
and environments. The presence of “base-off” cobalamin in ATR
and corrinoid iron-sulfur proteins has led to increased interest
in the electronic structure of this cofactor and suggests that an
investigation of base-off cobalamins may contribute to both an
understanding of the intrinsic electronic structure of these
important coenzymes and to the ways in which the environment
can influence the reactivity of the critical C-Co bond. The
transition from base-on to base-off can be achieved by lowering
the pH of the sample and is characterized by a blue-shift of the
dominant absorption feature by ca. 55 nm, from 525 to 459 nm
in AdoCbl (Figure 2). A similar blue-shift is observed for MeCbl
and other alkylcobalamins. In this paper, we present transient
absorption studies of AdoCbl, MeCbl, and PrCbl at pH 2 where
the dimethylbenzimidazole base is protonated and the cobalamin
is in a base-off configuration.
Experimental Methods
Adenosylcobalamin and methylcobalamin were obtained from
Sigma-Aldrich and used without further purification. n-Propyl-
cobalamin was synthesized and purified according to literature
methods as described previously.34 The aqueous solutions were
prepared and studied under anaerobic conditions at all times.
Doubly distilled water was deoxygenated by bubbling nitrogen
through it for a minimum of 1 h and added to the solid
cobalamin samples immediately prior to the spectroscopic
measurements. The sample concentrations used were 0.8 mM
for adenosylcobalamin, 2.1 mM for methylcobalamin, and 1.5
mM for n-propylcobalamin. To prepare the cobalamins in their
base-off form, the solution pH value was adjusted to 2 with the
addition of a small volume of deoxygenated HCl (2 M standard
Kozlowski and co-workers have applied TD-DFT methods
to explore photochemical bond homolysis in MeCbl and
EtCbl.17,20 In accord with the finding that the BP86 functional
does a better job of modeling the C-Co bond, this functional
was used to study the electronic state manifold along the reaction
3
coordinate. These calculations identify a dissociative (σCo-C
f σ*Co-C) state that drops in energy as the C-Co bond

12400 J. Phys. Chem. B, Vol. 114, No. 38, 2010
Peng et al.
solution, Sigma-Aldrich). A nitrogen atmosphere of positive
pressure was maintained above the sample solutions over the
course of the experiments to prevent oxygen dissolution. The
samples were kept in a reservoir and circulated through a flow
cell of 1 mm optical path length (0.5 mm for MeCbl) to refresh
the sample volume between laser pulses.
Ultrafast transient absorption measurements with 405-410
nm pump and white light continuum probe were performed
using a 1 kHz Ti:sapphire laser system described previously.36
Briefly, output from a self-mode-locked Ti:sapphire oscillator
was amplified with a multipass amplifier and compressed into
70 fs pulses centered at approximately 810-820 nm, with 250
µJ per pulse. A beam splitter was used to reflect 80% of the
pulse energy to be frequency doubled in a 500 µm type I
ꢀ-barium borate (BBO) crystal, generating pump pulses centered
at 405-410 nm with 6 µJ per pulse energy. The remaining 20%
of the amplified pulse energy was focused into 5 mm of flowing
ethylene glycol to generate a visible white light continuum. With
a spectrum extending from 400 nm to beyond 800 nm, the white
light continuum served as the broadband probe. For the
measurement of MeCbl photolysis, the continuum was generated
in a translating piece of CaF2, producing a continuum extending
from 340 nm to beyond 800 nm. The probe pulses were delayed
with respect to the pump pulses by a computer-controlled
motorized translation stage; the maximum delay was 1 ns. The
pump and probe beams were focused into a 200 µm spot size
and made to overlap inside the sample volume, near their
respective beam waists. In all measurements, the polarization
of the pump beam was set at magic angle (54.7°) with respect
to the polarization of the probe beam to ensure that only isotropic
responses were measured. A variable neutral density filter was
used to adjust the pump intensity for sample excitation. After
passing through the sample, the probe beam was coupled into
Figure 3. Change in absorption following excitation of AdoCbl at
pH 2. Left: The transient spectra at 5 ps (blue) and 600 ps (red) are
compared with the steady state spectrum in the top panel. The bottom
panel contains a contour plot of the absorption change between 425
and 650 nm over the first 100 ps. Right: The top graph shows sample
traces (solid lines) with exponential fits (dashed lines). The residuals
are also included in the plot. The bottom graph shows the decay
associated difference spectra (DAD spectra) obtained by plotting the
amplitude of each exponential decay component as a function of
wavelength. The red line is the long-lived component measured at 600
ps, the same as shown in the upper left-hand plot.
obtained at pump intensities where linear excitation dominates
the observed transient absorption signal.
The steady state spectrum of base-off cob(II)alamin was
measured by photolysis of AdoCbl in the presence of the radical
scavenger TEMPO as reported previously,34,35 followed by
addition of acid to drop the pH of the sample. The spectrum
was also measured by photolysis of AdoCbl directly at pH 2 in
the presence of the radical scavenger. The same product
spectrum is obtained by each method, as shown in Figure 2,
although the shoulder around 540 nm varies somewhat from
scan to scan and may represent the contribution of an oxidation
product. UV-visible absorption spectra are reported by Stich
et al. for cob(II)inamide prepared by KCOOH reduction of
adenosylcobinamide (a cobalamin derivative that lacks the
nucleotide loop and the dimethylbenzimidazole base).15 These
spectra are consistent with the possibility that the shoulder
around 540 nm represents an oxidation product or incomplete
dissociation. The shoulder may also arise from a different
conformational placement of the phosphate linkage and di-
methylbenzimidazole group absent in the cobinamide.
the entrance slit of
a photodiode array spectrometer
(BWTEK741E-512, for AdoCbl and PrCbl) or a CCD based
spectrometer (AvaSpec-2048-USB2, for MeCbl) with an optical
fiber. A 1 mm BG39 Schott glass filter was used to attenuate
the excessive near-IR component of the transmitted probe to
prevent detector saturation. A mechanical chopper synchronized
to the 1 kHz reference was placed in the pump beam and set to
operate at 100 Hz, providing five-pulse pump-on/pump-off
modulations. Taking the common log of the transmitted probe
intensity ratio then gave the difference absorption spectra. A
difference absorption spectrum recorded at one delay step was
computed from averaging 800 pairs of pump-on/pump-off
measurements. The data from four repeated measurements were
averaged together to give the frequency resolved transient
absorption spectra reported below.
Results
The photochemistry of alkylcobalamins may contain contri-
butions arising from multiphoton excitation of the cofactor.
Because the magnitude of the difference spectrum at long times
(>200 ps) following excitation of the base-off compounds is
quite small (see below), it was important to investigate this
power dependence. The variable neutral density filter in the
pump arm was used to vary the pump intensity over a factor of
4 from 0.5 to 2 µJ/pulse. The 600 ps scans in base-off AdoCbl
exhibit a structured peak at 470 nm with a linear dependence
on the pump intensity and a component with approximately
cubic intensity dependence and an almost monotonically
increasing intensity from 420 to 660 nm. A similar spectral
feature, increasing across the visible and peaking at wavelengths
longer than 700 nm, is observed following multiphoton excita-
tion of base-on MeCbl at 400 nm. The data reported below for
the photolysis of AdoCbl, MeCbl, and PrCbl at pH 2 were
A. Transient Absorption and Decay Associated Difference
Spectra. The transient absorption spectrum of AdoCbl at pH 2
is plotted in Figure 3. Excitation at 400 nm is followed by
formation of a deep bleach within a few picoseconds. The broad
bleach centered at ca. 480 nm indicates a blue-shift of the excited
state spectrum relative to the ground state spectrum. The bleach
recovers, leaving a small residual photoproduct spectrum at time
delays >200 ps. The data obtained every 3 nm were fit to a
sum of three exponential decay components, a fast component
of ca. 0.5 ps, and two well-defined components of 2.9 ( 0.24
and 60 ( 3 ps, with a small residual that does not decay on the
subnanosecond time scale. Additional components appear to be
present at early time, but analysis of the early time behavior is
complicated by contributions from coherent two-photon absorp-
tion (400 nm + continuum) and by cross-phase modulation
within the chirp of the continuum. Sample fits and the decay

Base-Off B12 Coenzymes and Analogues
J. Phys. Chem. B, Vol. 114, No. 38, 2010 12401
Figure 5. Change in absorption following excitation of MeCbl at pH
2. Left: The transient spectra at 5 ps (blue) and 500 ps (red) are
compared with the steady state spectrum in the top panel. The bottom
panel contains a contour plot of the absorption change between 425
and 650 nm over the first 100 ps. Right: The top graph shows sample
traces (solid lines) with exponential fits (dashed lines). The residuals
are also included in the plot. The kinetic traces run to 600 ps. The
bottom graph shows the decay associated difference spectra (DAD
spectra) obtained by plotting the amplitude of each exponential decay
component as a function of wavelength. The solid red line is the long-
lived component in the fit to the data.
Figure 4. Change in absorption following excitation of PrCbl at pH
2. Left: The transient spectra at 5 ps (blue) and 400 ps (red) are
compared with the steady state spectrum in the top panel. The bottom
panel contains a contour plot of the absorption change between 425
and 650 nm over the first 100 ps. Right: The top graph shows sample
traces (solid lines) with exponential fits (dashed lines). The residuals
are also included in the plot. The kinetic traces run to 400 ps. The
bottom graph shows the decay associated difference spectra (DAD
spectra) obtained by plotting the amplitude of each exponential decay
component as a function of wavelength. The solid red line is the long-
lived component in the fit to the data.
associated difference spectra (DAD spectra) defined by the
amplitudes of each component are plotted in Figure 3.
The transient absorption spectrum obtained following excita-
tion of propylcobalamin is significantly different. There is a net
bleaching of the ground state absorption at 510 nm but a net
increase in absorption from 425 to 480 nm. The data plotted in
Figure 4 were fit to a sum of four exponential decay components,
a fast component of ca. 0.25 ps and two well-defined compo-
nents of 2.0 ( 0.3 and 18 ( 2 ps. A fourth decay component
of 100 ( 40 ps was also required by the data. This component
has small amplitude, and the precise decay constant is not well-
defined by the data; a time constant anywhere from 60 to 150
ps provides a reasonable fit. A small residual signal correspond-
ing to photoproduct does not decay on a subnanosecond time
scale. This signal is larger than the corresponding signal
observed following excitation of AdoCbl. Sample fits at three
wavelengths and the DAD spectra defined by the amplitudes
of each component are plotted in Figure 4. The DAD spectra
are not affected much by the precise time constant of the ca.
100 ps component. The influence of this rate constant is
illustrated in additional plots contained in the Supporting
Information.
Figure 6. Left: Species associated difference spectra (SAD spectra)
calculated for base-off AdoCbl (top) and PrCbl (bottom) following
excitation at 400 nm given the model shown in the figure. Lifetimes
are shown for AdoCbl (PrCbl). Right: Species associated spectra (SA
spectra) calculated from the SAD spectra assuming excitation of 9%
of the molecules in the probed volume for AdoCbl and 3% of the
molecules in the probed volume for PrCbl.
The transient absorption spectrum obtained following excita-
tion of methylcobalamin is shown in Figure 5. The continuum
probe used for this experiment allowed measurement of the
transient spectra over a broader wavelength range. There is a
net bleaching of the ground state absorption at 500 nm, a net
increase in absorption around 410 nm, and a net bleaching again
at 375 nm. The data plotted in Figure 5 were fit to a sum of
three exponential decay components, a fast component of ca.
0.36 ps, and two well-defined components of 2.1 ( 0.4 and 47
( 4 ps. The fit to three exponentials provides a good global
picture of the dynamics but is not perfect. There is evidence
for a small amplitude relaxation component with wavelength
dependent rate constant. Sample fits at three wavelengths and
the DAD spectra defined by the amplitudes of each component
are plotted in Figure 5.
describe the data but provide little intuitive insight into the nature
of the excited electronic states. Species associated spectra
provide a better understanding but require a model to interpret
the observed spectral changes. The simplest model accounting
for the data involves stepwise progression through a series of
electronic or conformational states. Excitation results in rapid
production of population in an excited state A, the earliest state
clearly identified in the data. This population decays on a time
scale of 0.25-0.50 ps, proceeding through two distinguishable
intermediate states, B and C, to a final state D that persists for
times .400 ps. An additional state D′ is included for PrCbl,
reflecting a relaxation of D or an additional excited electronic
state. This scheme is illustrated in Figure 6. The species
associated difference spectra (SAD spectra) calculated from the
DAD spectra for AdoCbl and PrCbl are plotted in the left-hand
panel of Figure 6.
B. Construction of the Species Associated Spectra. The
decay associated difference spectra plotted in Figures 3-5

12402 J. Phys. Chem. B, Vol. 114, No. 38, 2010
Peng et al.
The SAD spectra provide some insight into the nature of the
excited state species, but the nature of these states can be seen
more clearly in the species associated excited state spectra. The
species associated spectra can be reconstructed from the
difference spectra given knowledge of the percentage of
molecules excited in the probe volume. For AdoCbl at pH 2,
the extinction coefficient at 405 nm is 7.4 × 103 M-1 cm-1
;
thus, the absorbance of a 1 mm path length sample of 8 × 10-4
M is 0.59 and 74% of the available photons will be absorbed
and 26% will be transmitted. The estimated pulse energy at the
sample was 0.9 µJ. At 4.9 × 10-19 J/photon, absorption of 74%
of incident photons will yield 1.4 × 1012 excited molecules.
The spot size was 0.2 mm in diameter, and the number of
molecules in the focal area was 1.5 × 1013. Thus, the fraction
excited is estimated at 1.4/15 ) 0.09. Similar calculations for
the measurements of PrCbl and MeCbl yield estimates for the
excited fraction of 0.03 and 0.075, respectively. These fractions
were used to reconstruct the species associated spectra in Figure
6. The reasonable limits for the error in these reconstructions
and details for the estimates for PrCbl and MeCbl are presented
in the Supporting Information.
Figure 7. Left: Species associated difference spectra (SAD spectra)
calculated for base-off MeCbl following excitation at 410 nm given
the model shown in Figure 6. Right: Species associated spectra (SA
spectra) calculated from the SAD spectra assuming excitation of 7.5%
of the molecules in the probed volume.
The species associated spectra in Figure 6 demonstrate that
following excitation at 400 nm AdoCbl exhibits a broad excited
state absorption red-shifted from the ground state spectrum. The
transition from A to B is correlated with a blue-shift of the
excited state absorption on a 0.5 ps time scale. The transition
from B to C is correlated with an additional blue-shift of the
spectrum. Comparison of the spectra corresponding to states B
and C suggests that the 2.9 ps transition represents a relaxation
or conformational change in the excited electronic state rather
than a transition from one electronic state to another.
Figure 8. Estimated spectra of the long-lived photoproducts produced
following excitation of PrCbl (green solid line, quantum yield ) 0.19),
AdoCbl (red dashed line, quantum yield ) 0.13), and MeCbl (light
blue line, quantum yield ) 0.65). The cob(II)alamin spectrum measured
at pH 2 is shown for comparison. The dotted line represents the room
temperature Co(II) cobinamide spectrum reported by Stich et al. in
Figure 2 of ref 15.
The evolution of the excited state spectrum over the first few
picoseconds provides a picture of the rapid internal conversion
and relaxation of the molecule in the lowest accessible electronic
excited state. There is no evidence for intersystem crossing or
bond dissociation on this time scale. Thus, the spectrum of state
C is assigned to the S1 f Sn transitions of base-off AdoCbl,
where the S1 state is the lowest excited singlet state and Sn
represents the manifold of higher-lying excited states accessible
from this state. The decay of this excited state on a time scale
of 60 ps yields a spectrum nearly identical to the initial ground
state spectrum. It is likely that this transition represents a
branching between formation of a long-lived product and return
to the ground state. The nature of the long-lived photoproduct
is discussed in greater detail below.
transition from B to C on a 2 ps time scale is accompanied by
a small decrease in the intensity of the absorption, predominantly
the 466 nm peak. The spectrum of state C is assigned to the S1
f Sn transitions of the lowest excited singlet state of base-off
MeCbl. The decay of this excited state on a time scale of 47 ps
yields a long-lived product with a spectrum peaking at 469 nm.
The spectrum of the long-lived photoproduct is stronger than
the corresponding transient in AdoCbl or PrCbl.
C. Identification of the Long-Lived Photoproduct as
Cob(II)alamin. At pH 2, when the compound is in a base-off
conformation, the difference spectrum observed at 400 ps in
PrCbl and 600 ps in AdoCbl and MeCbl is characterized by an
absorption peak around 470 nm, consistent with formation of a
base-off cob(II)alamin. The spectrum of the long-lived photo-
product can be estimated from the data presented above
assuming that a similar cob(II)alamin product is formed
following excitation of all three compounds. These spectra are
plotted in Figure 8. The photolysis product lacks the shoulder
at 540 nm observed for base-on and base-off cob(II)alamin
produced via room temperature photolysis. However, the
spectrum of the product, especially following excitation of PrCbl
and MeCbl, is in good agreement with the Co(II) cobinamide
spectrum reported by Stich et al.15 The agreement is quantitative
and qualitative; both the shape and the estimated extinction
coefficients agree. The spectrum observed following excitation
of AdoCbl differs somewhat around 540 nm, but this difference
spectrum (see Figure 3) is weak with a relatively small signal-
to-noise ratiosthe noise is magnified in Figure 8. From these
comparisons, we conclude that bond homolysis occurs on a time
scale of a few tens of picoseconds.
Excitation of PrCbl results in a broad excited state absorption
peaking to the blue of the probe window. This spectrum decays
faster than the corresponding species in AdoCbl, and the error
in the amplitude is larger. The transition from B to C is
accompanied by a blue-shift of the excited state spectrum and
a slight decrease in the intensity of the absorption. The spectrum
of state C is again assigned to the S1 f Sn transitions from the
lowest excited singlet state of base-off PrCbl. The decay of this
excited state on a time scale of 18 ps yields a spectrum similar
to the initial ground state spectrum. Again, it is likely that the
decay of the C state is accompanied by branching between
formation of a long-lived photoproduct and return to the ground
state. The small amplitude of the long-lived transient makes it
difficult to observe changes on a longer time scale for either
AdoCbl or PrCbl.
Excitation of MeCbl results in a similar excited state
progression (Figure 7), with a broad excited state absorption
decaying on a ca. 360 fs time scale to a more structured excited
state absorption with peaks around 535 and 466 nm. The

Base-Off B12 Coenzymes and Analogues
J. Phys. Chem. B, Vol. 114, No. 38, 2010 12403
The estimated quantum yield for bond homolysis is 0.12 (
0.06 following excitation of AdoCbl and 0.2 ( 0.1 following
excitation of PrCbl. In contrast, the estimated quantum yield
for bond homolysis in MeCbl is substantially larger at 0.65
(+0.15, -0.25). The error bars reflect uncertainties in the
measurements, including the uncertainty in the percentage of
molecules excited within the sample volume and uncertainty
in the extinction coefficients of the base-off spectra. These
quantum yields reflect the primary yield of the radical pair based
on the observed difference spectra between 400 and 600 ps.
The time scales for diffusive separation of the adenosyl and
propyl radicals from the cob(II)alamin in water at 20 °C, pH 7,
are 1.6 and 1.5 ns, respectively.37 Thus, the steady state quantum
yield may be influenced by geminate recombination of the
radical pair on a time scale longer then the measurements
reported here. There is no evidence for geminate recombination
in the data, but the signals are small enough that a small
amplitude nanosecond recombination cannot be ruled out.
The methyl radical diffuses significantly faster than the larger
adenosyl and n-propyl radicals. An effective lifetime of
130-400 ps is expected for the contact radical pair based on
measurements of recombination in the base-on compound and
on simple hydrodynamic calculations.37 In the data reported here
for base-off MeCbl, the transient absorption signal between 200
and 600 ps is constant. Geminate recombination does not occur
with any appreciable yield.
These quantum yields can be compared with the quantum
yields reported by Taylor et al. for steady state aerobic photolysis
of the base-off cobalamins at pH 1.27 Photolysis of base-off
AdoCbl (pH 1) resulted in a homolysis yield of ca. 0.02-0.04
across the spectrum from 250 to 470 nm. This is somewhat
lower than the estimate of 0.12 in the current study. Geminate
recombination of the radical pair may account for the discrep-
ancy. The photolysis yield following excitation of base-off PrCbl
at pH 1 was 0.26 ( 0.02 from 250 to 500 nm, in reasonable
agreement with the present measurement at 405 nm. The yield
for aerobic homolysis of MeCbl at pH 1 is 0.27 ( 0.02 from
470 to 350 nm, somewhat higher at shorter wavelengths and
lower at longer wavelengths. It appears that the primary yield
at 600 ps is substantially larger than the steady state quantum
yield. Geminate recombination will not account for the
discrepancy.
to π f π* transitions with vibrational progressions accounting
for the shoulders and subpeaks. More recently computational
and spectroscopic studies have called this into question, assign-
ing the major features to a complicated manifold of electronic
states.11-21,38 Accurate electronic structure calculations will be
able to account for the similarities and differences in the ground
and excited state spectra. Thus, these observations provide a
constraint for evaluating the calculations.
A. Electronic Structure and Excited State Spectra. Recent
TDDFT quantum chemical calculations by Kozlowski and
co-workers18,19 and Brunold and co-workers5,12 have explored
the electronic structure of base-off adenosyl and methyl cobi-
namides (AdoCbi and MeCbi). Most of these calculations have
examined the vertical transitions at the ground state equilibrium
geometry. In contrast, the transient absorption experiments probe
the excited state spectra as a function of conformational
relaxation and at the excited state minimum energy configura-
tion. Nonetheless, it is useful to consider the experimental results
in the context of these calculations. Koszlowski and co-workers
used B3LYP and BP86 functionals in TDDFT calculations of
the electronic transition of MeCbl, MeCbi, AdoCbl, and
AdoCbi.18,19 Calculations of AdoCbl and AdoCbi were also
performed using both functionals and a polarizable continuum
model to account for the influence of water solvation.18
In all of the calculations of base-on and base-off cobalamins,
the oscillator strength is carried by transitions involving π f
π* transitions of the corrin ring. The calculations using the
B3LYP functional do a better job of handling the π f π*
transitions than the calculations using the BP86 functional. As
a result, the B3LYP functional provides a prediction of the
absorption spectra. However, the BP86 functional is expected
to do a better job of modeling the d/π f π*/d transitions
sensitive to the axial ligation of the cobalt.18,19 Thus, it is useful
to consider the results of both methods. All of the calculations
for the cobinamides predict one or more states below the π f
π* states responsible for the absorption spectrum.
The B3LYP calculations of MeCbi predict that the lowest
singlet state, S1, contains contributions from the dyz + π, dxz +
π, and π + dyz f σ*(Co-C) configurations. The S2 state contains
contributions from the dxz + π f σ*, π + dy f dxy + π*, and
π + dz2 f π* configurations. Both of these states are lower in
energy than the dominant π f π* states carrying the oscillator
strength. The calculation using the BP86 functional predicts only
one state, with dominant dxy + π f π* character, below the
allowed π f π* states (Tables S4 and S5 of ref 19). Calculations
of AdoCbi show similar trends, with an Ado(π) f π*
configuration added to the mix (Tables S4 and S5 of ref 18). In
fact, the lowest state is dominated by the Ado(π) f π*
configuration in the calculation using the BP86 functional. When
the water solvent is approximated using the PCM model, the
Ado(π) f π* transitions move to higher energy, although
Ado(π*) mixes with σ*(Co-C) and contributes to the lowest
transitions in the calculation using the B3LYP functional (Tables
S11 and S12 of ref 18).
Discussion
The data presented above provide important new insights into
the electronic structure and reactivity of alkylcobalamins. The
mechanism for bond homolysis is modified by the presence or
absence of the nitrogenous axial ligand. The difference in
photochemistry reflects the influence of the axial ligand on the
electronic structure of the cobalamin and provides additional
insight into the role ligation and environment play in the
reactivity of the C-Co bond. Accurate electronic structure
calculations will be required to fully interpret the observed
photochemistry and photophysics of the cobalamin cofactors.
It is also noteworthy that the spectra of the lowest-lying
excited states populated following excitation of these three base-
off alkylcobalamins are remarkably similar to the spectra of
the parent ground state molecules. There is a very weak red
absorption tail observed for all three molecules, but the major
absorption features begin around 465 nm for both PrCbl and
AdoCbl (Figure 6). MeCbl appears to have a smaller band
around ca. 530 nm followed by a stronger transition at 465 nm
(Figure 7). Early models for the electronic structure of cobal-
amins assigned the major transitions in the absorption spectrum
It is dangerous to draw too many conclusions from these
calculations. They were performed at the ground state geometry
without allowing the excited state geometry to relax. In addition,
the methods are not yet well calibrated for the dark states
strongly influenced by the Co d atomic orbitals. Nonetheless, it
is suggestive that the lowest states are dominated by d orbital
excitations. It is also interesting that the σ*(Co-C) orbital plays
a significant role in the lowest transitions, at least in the B3LYP
calculations. It appears likely that the excited state spectra of
all three base-off compounds resemble the ground state spectra

12404 J. Phys. Chem. B, Vol. 114, No. 38, 2010
Peng et al.
because the π f π* transitions are modified only slightly by
the change in electronic configuration from the ground to S1
excited state. In contrast, the lowest state of the base-on
cobalamins is an allowed π f π* state in most of the
calculations.18,19 If the dominant configuration at the minimum
energy geometry of the excited state remains the π f π*
configuration, it will explain the more significant differences
in excited state spectra for most of the base-on compounds.36
The observed excited state absorption spectra provide constraints
to help validate future electronic structure calculations of
alkylcobalamins.
B. Photolysis Mechanism. The photolysis quantum yield in
base-off AdoCbl, PrCbl, and MeCbl is dominated by the
competition between bond homolysis and fast internal conver-
sion back to the ground state. The data presented above
demonstrate that photolysis of these alkylcobalamins results in
a rapid cascade through the excited state manifold producing
an excited state with a lifetime of 18-60 ps, presumably the S1
state (B and C in Figures 6 and 7). The population in this state
branches between bond homolysis, forming base-off cob-
(II)alamin and an alkyl radical, and recovery of the ground state
alkylcobalamin. The relative stability of base-off cobalamins
to photodissociation results from a dominant picosecond channel
for ground state recovery. The intrinsic rate constant for
nonradiative decay to the ground state is estimated from the
observed rate constants and estimated quantum yields at ca.
40-50 ns-1 for PrCbl, 13-16 ns-1 for AdoCbl, and 4-13 ns-1
for MeCbl.
white light source for excitation. To test the possible influence
of the wavelength-dependent photochemistry of MeCbl, Jones
et al. compared measurements carried out with a <400 nm filter
and in the absence of the filter. While the photolysis yield
reflected the wavelength dependence reported in other studies,
the magnetic field effect was unaffected by the filter (Figure
S5 of ref 25). From this, it was inferred that the initial radical
pair is produced in the singlet state independent of excitation
wavelength.
In contrast, the lack of geminate recombination in base-off
cobalamins is consistent with the formation of the geminate
3
radical pair in a triplet state, the (σCo-C f σ*Co-C) state, and
suggests possibilities for future studies, both experimental and
computational.
Finally, another extreme is seen in the base-on cob(III)alamins
with nonalkyl ligands. These compounds are photostable because
a channel for rapid internal conversion leads to ground state
recovery.39 The lifetime of the excited state is 1-7 ps (rate
constant 150-1000 ns-1) in room temperature aqueous solution,
with no permanent photoproduct formed. The nature of the axial
ligand in these cob(III)alamins opens a channel for internal
conversion an order of magnitude faster than the channel in
base-off alkylcobalamins. Calculations comparing the photolysis
of base-off and base-on cobalamins, and comparing both with
the rapid internal conversion in nonalkylcobalamins, may shed
new light on the influence of axial ligation on the electronic
structure.
Summary and Conclusion
In contrast, the base-on alkylcobalamins exhibit no ground
state recovery on a subnanosecond time scale. The photolysis
quantum yield in base-on AdoCbl and PrCbl at neutral pH is
determined by the competition between primary geminate
recombination of the radical pair and diffusive separation of
the radicals.29,34 This geminate recombination behaves as
expected as a function of both temperature and solvent viscos-
ity.37 The only clear evidence for ground state recovery via
internal conversion is observed following excitation of meth-
ylcobalamin (MeCbl). At room temperature, the excited state
decays on a 1 ns time scale with the quantum yield for ground
state recovery ∼0.85. Thus, the rate constant for internal
conversion is ∼0.85 ns-1. The rate constant for internal
conversion in all other alkylcobalamins investigated (ethyl,
propyl, adenosyl, and hexylnitrile) is no larger than the rate
constant in MeCbl.
The relative stability of base-off cobalamins to photodisso-
ciation arises from a dominant picosecond channel for ground
state recovery. The photolysis yield is controlled by competition
between internal conversion and bond homolysis. No significant
recombination of the geminate radical pair is observed. This is
in distinct contrast to the base-on cobalamins where the
photolysis yield is controlled to a large extent by competition
between geminate recombination and diffusive separation of the
geminate radical pair. The nature of the changes in electronic
structure of the base-off alkylcobalamins, opening a channel
for rapid internal conversion and nonradiative decay and
reducing geminate recombination, presents a challenge for
quantum chemical simulations of the C-Co bond and will shed
light on the mechanism available for the control of the reactivity
of this critical enzymatic site.
Replacement of the bottom nitrogenous axial ligand in MeCbl,
PrCbl, and AdoCbl with a water molecule changes the electronic
structure and opens a channel for rapid internal conversion. The
rate constant for internal conversion increases ∼10-fold in
MeCbl and 10-50-fold or more in AdoCbl and PrCbl. The
nature of this channel is not clear from the electronic structure
calculations performed to date. Elucidation awaits calculations
that include the influence of geometry relaxation in the excited
state.
Acknowledgment. This work is supported in part by the NSF
through grant CHE-0718219. Y.Y. was supported by the
summer China-USA REU program.
Supporting Information Available: Plots illustrating the
influence of the magnitude of the long time constant in the
analysis of PrCbl and the limits for acceptable reconstruction
of the species associated spectra. The estimates for excitation
percentages of PrCbl and MeCbl are worked out. This material
is available free of charge via the Internet at http://pubs.acs.org.
Of equal significance, the absence of a prominent geminate
recombination component reveals a change in the intrinsic
recombination rate for the geminate radical pair. Kozlowski and
co-workers reported TDDFT calculations predicting that pho-
tolysis of base-on cobalamins is mediated by the presence of a
3(σCo-C f σ*Co-C) state. This state drops in energy as the C-Co
bond length increases. However, production of a triplet radical
pair seems inconsistent with the magnetic field effect reported
by Jones et al.25 and is hard to reconcile with the prominent
geminate recombination component in the transient absorption
data.37 The magnetic field effect experiments used a broad band
References and Notes
(1) Banerjee, R.; Ragsdale, S. W. Annu. ReV. Biochem. 2003, 72, 209–
247.
(2) Matthews, R. G.; Koutmos, M.; Datta, S. Curr. Opin. Struct. Biol.
2008, 18, 658–666.
(3) Ludwig, M. L.; Matthews, R. G. Annu. ReV. Biochem. 1997, 66,
269–313.
(4) Marsh, E. N. G. Essays Biochem. 1999, 34, 139–154.
(5) Stich, T. A.; Seravalli, J.; Venkateshrao, S.; Spiro, T. G.; Ragsdale,
S. W.; Brunold, T. C. J. Am. Chem. Soc. 2006, 128, 5010–5020.

Base-Off B12 Coenzymes and Analogues
J. Phys. Chem. B, Vol. 114, No. 38, 2010 12405
(6) Seravalli, J.; Brown, K. L.; Ragsdale, S. W. J. Am. Chem. Soc.
2001, 123, 1786–1787.
(25) Jones, A. R.; Woodward, J. R.; Scrutton, N. S. J. Am. Chem. Soc.
2009, 131, 17246–17253.
(7) Ragsdale, S. W.; Lindahl, P. A.; Munck, E. J. Biol. Chem. 1987,
262, 14289–14297.
(8) Padovani, D.; Banerjee, R. Biochemistry 2009, 48, 5350–5357.
(9) Yamanishi, M.; Labunska, T.; Banerjee, R. J. Am. Chem. Soc. 2005,
127, 526–527.
(10) Stich, T. A.; Yamanishi, M.; Banerjee, R.; Brunold, T. C. J. Am.
Chem. Soc. 2005, 127, 7660–7661.
(11) Brunold, T. C.; Conrad, K. S.; Liptak, M. D.; Park, K. Coord. Chem.
ReV. 2009, 253, 779–794.
(12) Stich, T. A.; Brooks, A. J.; Buan, N. R.; Brunold, T. C. J. Am.
Chem. Soc. 2003, 125, 5897–5914.
(13) Stich, T. A.; Buan, N. R.; Escalante-Semerena, J. C.; Brunold, T. C.
J. Am. Chem. Soc. 2005, 127, 8710–8719.
(14) Brooks, A. J.; Fox, C. C.; Marsh, E. N. G.; Vlasie, M.; Banerjee,
R.; Brunold, T. C. Biochemistry 2005, 44, 15167–15181.
(15) Stich, T. A.; Buan, N. R.; Brunold, T. C. J. Am. Chem. Soc. 2004,
126, 9735–9749.
(26) Taoka, S.; Padmakumar, R.; Grissom, C. B.; Banerjee, R. Bioelec-
tromagnetics 1997, 18, 506–513.
(27) Taylor, R. T.; Smucker, L.; Hanna, M. L.; Gill, J. Arch. Biochem.
Biophys. 1973, 156, 521–533.
(28) Walker, L. A., II; Shiang, J. J.; Anderson, N. A.; Pullen, S. H.;
Sension, R. J. J. Am. Chem. Soc. 1998, 120, 7286–7292.
(29) Yoder, L. M.; Cole, A. G.; Walker, L. A., II; Sension, R. J. J.
Phys. Chem. B 2001, 105, 12180–12188.
(30) Sension, R. J.; Cole, A. G.; Harris, A. D.; Fox, C. C.; Woodbury,
N. W.; Lin, S.; Marsh, E. N. G. J. Am. Chem. Soc. 2004, 126, 1598–1599.
(31) Sension, R. J.; Harris, D. A.; Stickrath, A.; Cole, A. G.; Fox, C. C.;
Marsh, E. N. G. J. Phys. Chem. B 2005, 109, 18146–18152.
(32) Walker, L. A., II; Jarrett, J. T.; Anderson, N. A.; Pullen, S. H.;
Matthews, R. G.; Sension, R. J. J. Am. Chem. Soc. 1998, 120, 3597–3603.
(33) Shiang, J. J.; Walker, L. A., II; Anderson, N. A.; Cole, A. G.;
Sension, R. J. J. Phys. Chem. B 1999, 103, 10532–10539.
(34) Cole, A. G.; Yoder, L. M.; Shiang, J. J.; Anderson, N. A.; Walker,
L. A., II; Banaszak Holl, M. M.; Sension, R. J. J. Am. Chem. Soc. 2002,
124, 434–441.
(16) Brooks, A. J.; Vlasie, M.; Banerjee, R.; Brunold, T. C. J. Am. Chem.
Soc. 2004, 126, 8167–8180.
(17) Lodowski, P.; Jaworska, M.; Andruniow, T.; Kumar, M.; Ko-
zlowski, P. M. J. Phys. Chem. B 2009, 113, 6898–6909.
(18) Andruniow, T.; Jaworska, M.; Lodowski, P.; Zgierski, M. Z.; Dreos,
R.; Randaccio, L.; Kozlowski, P. M. J. Chem. Phys. 2009, 131.
(19) Andrunio´w, T.; Jaworska, M.; Lodowski, P.; Zgierski, M. Z.; Dreos,
R.; Randaccio, L.; Kozlowski, P. M. J. Chem. Phys. 2008, 129, 085101.
(20) Jaworska, M.; Lodowski, P.; Andruniow, T.; Kozlowski, P. M. J.
Phys. Chem. B 2007, 111, 2419–2422.
(21) Kuta, J.; Patchkovskii, S.; Zgierski, M. Z.; Kozlowski, P. M.
J. Comput. Chem. 2006, 27, 1429–1437.
(22) Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. Soc. 1993, 115,
12152–12157.
(35) Sension, R. J.; Harris, D. A.; Cole, A. G. J. Phys. Chem. B 2005,
109, 21954–21962.
(36) Harris, D. A.; Stickrath, A. B.; Carroll, E. C.; Sension, R. J. J. Am.
Chem. Soc. 2007, 129, 7578–7585.
(37) Stickrath, A.; Carroll, E. C.; Dai, X.; Harris, D. A.; Rury, A.; Smith,
B.; Tang, K.-C.; Wert, J.; Sension, R. J. J. Phys. Chem. A 2009, 113, 8513–
8522.
(38) Kozlowski, P. M.; Andruniow, T.; Jarzecki, A. A.; Zgierski, M. Z.;
Spiro, T. G. Inorg. Chem. 2006, 45, 5585–5590.
(39) Shiang, J. J.; Cole, A. G.; Sension, R. J.; Hang, K.; Weng, Y.;
Trommel, J. S.; Marzilli, L. G.; Lian, T. J. Am. Chem. Soc. 2006, 128,
801–808.
(23) Grissom, C. B. Chem. ReV. 1995, 95, 3–24.
(24) Hung, R. R.; Grabowski, J. J. J. Am. Chem. Soc. 1999, 121, 1359–
1364.
JP104641U