How an Enzyme Tames Reactive Intermediates
A R T I C L E S
(Supporting Information), as does Aiso. Both Aiso and Tloc are of
the same sign (positive or negative), with well-defined relative
magnitudes (Supporting Information).
The decomposition of T into local and nonlocal terms
•
(
Supporting Information) shows that Tn-loc > Tloc for 3(SLys )
•
and 3(DHLys ). As a result, T has the positive sign of Tn-loc.
The finding that Aiso and T have the same sign thus shows that
Aiso, and hence Tloc, are also positive. For 4(Lys ), Tn-loc < Tloc,
•
so it is possible that Aiso, Tloc, and T could all be negative.
However, we see no physical reason for Aiso to have the opposite
•
sign in 4(Lys ), and we drop this option.
The large isotropic 13C couplings indicate that there is
13
significant spin delocalization onto the 5′- CH3 probe from the
substrate-derived radical, which implies that, in each intermedi-
ate, the 5′-methyl of the 5′-Ado generated by H-atom transfer
1
3
to the 5′- CH2 radical of state 2 is in contact with the substrate
radical created by this reaction. The distance from the radical
1
3
spin to the C, as calculated from Tn-loc and eq 4, is of limited
value as the point-dipole approximation breaks down at short
1
3
•
•
•
Figure 3. 35 GHz C ENDOR of 3(SLys ), 4(â-Lys ), and 3(DHLys ).
•
2
2
distances, but the value obtained, r ≈ 2.4 Å for 4(Lys ) and
Inset: H ENDOR from sample with DHLys and [5′,5′- H2]-SAM. Baseline
from unlabeled sample has been subtracted. Spectra collected at peak of
radical EPR spectrum in each sample (g ) 2.002). Simulations shown in
blue below spectra. Experimental conditions: T ) 2 K; repetition rate )
•
3(SLys ), is of heuristic value and supports the conclusion that
13
the CH3-Ado is at least in van der Waals contact with the
•
25
•
•
•
13
radical (Supporting Information). For 3(DHLys ), analysis
1
00 Hz for 3(SLys ), 200 Hz for 4(â-Lys ) and 3(DHLys ) C, and 50 Hz
2 13
•
•
for 3(DHLys ) H; RF pulse length ) 60 µs. For 3(SLys ) C Davies pulse
requires that one take into account the 50/50 partitioning of the
sequence, MW pulse lengths ) 80, 40, and 80 ns, νMW ) 34.821 GHz; for
spin density onto the â- and δ-carbons. Such an analysis yields
•
4
(â-Lys ) Davies pulse sequence, MW pulse lengths ) 80, 40, and 80 ns,
13
a distance r( C-Câ,δ) ≈ 3.4 Å, again supporting the conclusion.
•
13
νMW ) 34.711 GHz; for 3(DHLys ) C ReMims pulse sequence, MW pulse
•
2
13
length ) 32 ns, τ ) 248 ns, νMW ) 34.691 GHz; for 3(DHLys ) H Mims
pulse sequence, MW pulse length ) 52 ns, τ ) 452 ns, νMW ) 34.682
The magnitude of the C coupling correlates with the extent
13
of orbital overlap between the radical and the 5′- CH3-Ado:
•
GHz. Simulation parameters: for 3(SLys ), T ) 2.5 MHz, Aiso ) 12.7 MHz,
•
the contact thus must be better in 4(Lys ) (Aiso ) 18.5 MHz)
•
ENDOR line width ) 1.0 MHz; for 4(â-Lys ), T ) 2.9 MHz, Aiso ) 18.5
•
•
than in 3(Slys ) (Aiso ) 12.7 MHz). The isotropic coupling for
MHz, ENDOR line width ) 1.8 MHz; for 3(DHLys ), T ) 0.6 MHz, Aiso
1
3
•
)
2 MHz, ENDOR line width ) 0.3 MHz, Mims suppression simulated
5′- CH3-Ado of 3(DHLys ), Aiso ) 2 MHz, is noticeably smaller
with τ ) 200 ns.
26
than for the other two intermediates. However, this coupling
nonetheless is large enough to indicate that the 5′- CH3-Ado
of 3(DHLys ) also is in van der Waals contact with its radical.
13
coupling and also shows the ν- peak (eq 1). We have simulated
the spectra (Figure 3) and found that each is describable by a
hyperfine tensor that is dominated by the isotropic hyperfine
coupling, Aiso, and accompanied by an axial anisotropic inter-
action, T ) [-T,-T,+2T]. The splitting between the maximum-
intensity (perpendicular) feature of the two branches of such
spectra is A⊥ ) (Aiso - T), the “parallel” splitting is A| ) (Aiso
2T), and the width of an individual branch is 3T/2. The
experimental hyperfine tensors give Aiso ) +18.5 MHz, T )
2.9 MHz for 4(Lys ), Aiso ) +12.7 MHz, T ) +2.5 MHz for
(SLys ), and Aiso ) +2.0 MHz, T ) +0.6 MHz for 3(DHLys ).
The shapes of the patterns show that Aiso and T have the same
sign; in the next paragraph we justify the choice of the positive
sign.
The anisotropic interaction is the sum of two axial compo-
nents. One is a tensor, Tn-loc, that arises from the dipolar
interaction between the 5′- CH3 nuclear spin and the electron
spin of the radical. It is characterized by a parameter, Tn-loc,
which is necessarily positive; in the point-dipole approximation
it is related to the electron-nuclear distance through the
expression
•
We suggest that Aiso is reduced primarily because the close
•
intermolecular contact involves the intervening γ-CH of DHLys ,
1
1
13
which carries negligible spin density; the smaller Aiso( C)
•
measured for the interaction with 3(DHLys ) may further reflect
poorer orbital overlap. As illustrated by Scheme 3, in the case
•
of 3(DHLys ), formation of the allyl radical significantly distorts
+
the carbon backbone relative to the natural substrate radical,
•
R-Lys .
•
+
3
2
•
•
The 5′-methylene of SAM also was deuterated (5′-C H2), and
H spectra were collected from 3(SLys ) and 3(DHLys ); the
2
•
•
•
2
EPR spectrum of 4(Lys ) was too weak to give satisfactory H
spectra. The 3(DHLys ) intermediate gives the H spectrum
•
2
shown in Figure 3, after subtraction of background signals from
1
4
∆
m ) (2 transitions of N. The spectrum shows a pair of
13
broad outer features whose maximum splitting is ∼0.8 MHz
and a pair of sharper inner features split by 0.3 MHz. The outer
features must be associated with a relatively strongly coupled
2
H. The inner features could be reasonably interpreted in either
2
of two ways: as a signal from more weakly coupled H or as
part of the broader signal. In either case, the well-defined
1/3
2
1
features of the spectrum suggest that the 5′-C H2 H-Ado group
r(Fe-N) ) [g â g â /Tn-loc]
(4)
e
e N N
(
24) Weil, J. A.; Bolton, J. R.; Wertz, J. E. Electron Paramagnetic Resonance:
Elementary Theory and Practical Applications; John Wiley & Sons: New
York, 1994.
where the constants have their usual meanings and the nuclear
24
g factors, gN, have previously been tabulated. The second
13
(25) DeRose, V. J.; Liu, K. E.; Lippard, S. J.; Hoffman, B. M. J. Am. Chem.
Soc. 1996, 118, 121-134.
(26) Although much larger than that to the methyl group of Met in this state.
tensor, Tloc, arises from interaction of the C with spin density
3
delocalized into the sp orbital of a methyl C-H/D bond
J. AM. CHEM. SOC.
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VOL. 128, NO. 31, 2006 10149