A R T I C L E S
Jones et al.
There are a range of publications on the dynamic spin
chemistry of AdoCbl, including: MFE in AdoCbl photolysis
AdoCbl 20 µM, determined spectrophotometrically at 525 nm using ꢀ
-
1
-1
)
8.0 mM cm ; and 2AE 30 mM. An AdoCbl concentration of 20
26
µM was used in this case, compared to 12 µM previously, because the
concentration ratio (∼1.7) corresponds to the ratio between reaction
cell optical pathlengths in this (10 mm) and the original study (17 mm).
Under safe-light, reagent solutions were passed through a 0.45 µm filter
on loading into the drive syringes: syringe 1, EAL apoenzyme; syringe
in solution; calculated and modeled MFE in AdoCbl-dependent
2
7,28
29
30,31
enzymes;
CIDNP and CIDEP
on AdoCbl, model
compounds, and analogues. The MFE in Vmax/Km has been
3
2
reproduced in collaboration with the original authors, and a
heavy atom effect was also observed in these parameters for
2
, AdoCbl and 2AE.
3
3
EAL. New stopped-flow data will now be presented for EAL
that challenge the relevance of the original stopped-flow
methodology to studying MFE and offer a new protocol that in
turn questions the established wisdom on the proposed magneti-
cally sensitive step in this system. These results have wider
implications for the catalytic power of AdoCbl-dependent
enzymes in terms of stabilization of the RP against geminate
recombination relative to free coenzyme.
All experiments were conducted under safe-light at 25 °C. Absor-
bance measurements were acquired over a linear time-base using a
25
single wavelength of 525 nm (which corresponds to the most
significant absorbance change on C-Co homolysis, see Supporting
Information) and a photomultiplier tube (PMT). The homogeneous MF
(30, 50, and 80 mT) was applied to the sample position throughout the
data acquisition period and generated by mounting pairs of rare-earth
permanent magnets in opposite, unoccupied light-guide ports in the
cell block. The first 5-6 shots were discarded on every occasion. Each
experiment comprised 6-7 field-on/field-off data acquisition pairs (the
order of which was randomized) and was repeated several times for
every MF data point.
Experimental Procedures
Materials. The plasmid, pET-SEAL, encoding the small (32.0 kDa)
and large (49.1 kDa) subunits of EAL from Salmonella typhimurium
1.2. AdoCbl Binding Studies. To assess the rate of cofactor binding
(
kindly donated by Prof. George Reed), was expressed in Escherichia
3
4
to the apoenzyme under the same conditions as above, but in the absence
of an applied MF, EAL was rapidly mixed with varying concentrations
of AdoCbl and absorbance measurements acquired at 555 nm (see
Supporting Information). Reagent solutions were prepared in B3: EAL
coli and purified as described previously. AdoCbl (coenzyme B12
,
>
98% Sigma), 2AE (ethanolamine, >98%, Sigma), 2AP (S-(+)-2-
2
amino-1-propanol, >98%, Aldrich), and H
98%, Cambridge Isotopes) were all used as purchased.
Stopped-Flow Spectrophotometry. Two mixing regimes were
4 4
-2AE (D -ethanolamine,
>
∼
13 µM active-sites; and AdoCbl 50, 75, 100, 125, and 150 µM. A
similar experiment was then carried out with 2AE alongside AdoCbl
i.e., apoenzyme mixing) and absorbance measurements acquired at
25 nm. Reagent solutions were prepared in B3: EAL ∼13 µM active-
sites; AdoCbl 20, 37.5, 50, 75, 100, and 125 µM; and 2AE 30 mM.
.1. Holoenzyme Pre-Steady-State Kinetic and MFE Studies.
employed for stopped-flow studies with EAL. In the first instance, an
EAL apoenzyme solution (protein only) is loaded into one drive syringe,
with a solution mixture of AdoCbl and substrate in the second.
Alternatively, EAL apoenzyme and AdoCbl cofactor are incubated as
the holoenzyme in the same drive syringe prior to rapid mixing with a
solution of just the substrate. For a full justification of the different
protocols, refer to the Results and Discussion section. Construction and
testing of the dedicated magnetic field effect stopped-flow spectro-
photometer (MFESFS) in use throughout these studies is detailed in
the supporting information of ref 18. EAL begins to denature within
approximately 2 h of thawing, even when kept on ice. Care was
therefore taken to prepare solutions and carry out experiments well
within this time. No benefit was apparent when utilizing degassed (see
Supporting Information) reagent solutions. All comparisons made are
(
5
2
25
Previous stopped-flow experiments with EAL have successfully used
a simpler buffer solution than described above: 20 mM Hepes/NaOH,
pH 7.5 (“B20”). Reagent solutions were therefore prepared in B20: EAL
1
0-13 µM active-sites (consistent for each substrate); AdoCbl 20 µM;
2
4
and substrate (2AE, H -2AE, or 2AP) 2.5 mM. The general experi-
mental procedure was conducted as for the apoenzyme mixing regime,
with the thermostatic water bath at 5 or 25 °C depending on
requirements, and syringes loaded as follows: syringe 1, EAL
holoenzyme; syringe 2, substrate. Absorbance measurements were
acquired over linear and split (an equal number of acquisition points
distributed over two unequal time periods) time bases at 525 nm. A
Helmholtz pair mounted upon the cell block of the MFESFS and a
pulsed power supply (both constructed in-house) were used to generate
an adjustable, homogeneous MF (10 and 30 mT) at the sample position
throughout data acquisition. Due to the restrictions of this apparatus,
rare-earth permanent magnets were mounted as above for the 50 and
80 mT field points. Each experiment comprised 6-7 field-on/field-off
data acquisition pairs as above and was repeated several times for every
MF data point.
1
9
with the previous EAL stopped-flow MFE study.
.1. Apoenzyme Pre-Steady-State Kinetic and MFE Studies. The
1
apoenzyme mixing regime is the protocol adopted by the original MFE
stopped-flow study, and therefore, this reproduction adhered to the same
conditions wherever possible and appropriate. The buffer solution used
previously contained: 100 mM Hepes/NaOH, pH 7.45; 10 mM KCl;
1
0 mM urea; 5 mM dithiothreitol (DTT); and 10% glycerol; (designated
34
buffer “B4”). However, the enzyme purification process employed is
based on the limited solubility of the EAL protein near neutral pH in
buffers also containing glycerol. Consequently, a second buffer solution
2
.2. Holoenzyme Post-Steady-State MFE Studies. After the
(“B3”) was also prepared which has the same composition as B4, but
substrate has been exhausted by the enzyme turnover, a second transient
may be observed. This has been attributed to RP recombination of the
C-Co bond in AdoCbl25 and was also monitored for MF sensitivity.
Reagent solutions were prepared in B20: EAL ∼13 µM active-sites;
AdoCbl 20 µM; and 2AE 2.5 mM. The general experimental procedure
was conducted as before, with absorbance measurements acquired at
excluding glycerol. All solutions were prepared separately in both B4
and B3, with identical MFESFS investigations carried out in each case.
All concentrations quoted are post-mixing: EAL ∼10 µM active-sites;
(
(
(
25) Bandarian, V.; Reed, G. H. Biochemistry 2000, 39, 12069.
26) Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. Soc. 1993, 115, 12152.
27) Canfield, J. M.; Belford, R. L.; Debrunner, P. G. Mol. Phys. 1996, 89,
2
5 °C over a linear time-base at 525 nm. A magnetic field of 80 mT
8
89.
(
28) Eichwald, C.; Walleczek, J. Biophys. J. 1996, 71, 623.
29) Kruppa, A. I.; Taraban, M. B.; Leshina, T. V.; Natarajan, E.; Grissom, C.
B. Inorg. Chem. 1997, 36, 758.
was generated with rare-earth permanent magnets as above. This field
was applied to alternate shots for the entire 10 s data acquisition, which,
under the conditions, was sufficient time to ensure exposure of the full
steady-state turnover and post-steady-state transient.
3. MFE Studies on the Anaerobic Continuous-Wave Photolysis
of AdoCbl. The relevance of MFE to AdoCbl-dependent enzymes was
based on the observation of field sensitivity in the net quantum yield
of C-Co photolysis in the free cofactor.26 We have reinvestigated the
(
(30) Sakaguchi, Y.; Hayashi, H.; I’Haya, Y. J. J. Phys. Chem. 1990, 94, 291.
31) Bussandri, A. P.; Kiarie, C. W.; Van, Willigen, H. Res. Chem. Intermed.
(
2
002, 28, 697.
(
(
(
32) Taoka, S.; Padmakuma, R.; Grissom, C. B.; Banerjee, R. Bioelectromag-
netics 1997, 18, 506.
33) Anderson, M. A.; Xu, Y.; Grissom, C. B. J. Am. Chem. Soc. 2001, 123,
6
720.
34) Bandarian, V.; Reed, G. H. Biochemistry 1999, 38, 12394.
1
5720 J. AM. CHEM. SOC. VOL. 129, NO. 50, 2007
9