and 1.40, which is indicative of a His–Fe–Met
coordination. As the protein becomes oxidized
with time, large changes are evident in the EPR
spectra. These spectra evolve with primary
features of g values at 2.51, 2.20, and 1.86 that
are consistent with a His–Fe–Tyr coordination.
Also present as a minor component is a highspin species with g values of 6.88 and 5.07.
The combination of these spectroscopic studies
leads to a model of the protein as having three
states (Figure 16.25). Two of these states are
active states: the initial protein conformation that binds the hydroxylamine and the
resulting oxidized state. The third state is an
inactive state that is stable for a prolonged time
but can be returned to the active state by other
regulated proteins.
RESEARCH DIRECTION: RIBONUCLEOTIDE REDUCTASE
A critical step of DNA biosynthesis is the conversion of nucleotides into
deoxynucleotides, which is catalyzed by several classes of enzyme known
collectively as ribonucleotide reductase. Many of these enzymes, class I
ribonucleotide reductases, possess a characteristic EPR spectrum centered
around g = 2.00 with a pronounced hyperfine interaction (Figure 16.26).
Although these enzymes contain two iron atoms that form a diferric
cluster, the EPR spectrum does not arise from the metal cofactors. Rather,
the spectrum arises from a third non-metal
cofactor, a tyrosine radical.
The existence of amino acid radicals was
first discovered in proteins in the 1970s and
they have now been identified in a number of different enzymes (Stubbe & van der
Dork 1998; Stubbe 2003). In each case, the
amino acid radical can be identified primarily by the EPR spectrum, which should
be centered near g = 2.00 and have hyperfine structure. In some cases, the radicals
are transient species, but in the case of
ribonucleotide reductase the tyrosine radical is stable. Surprisingly, the tyrosine
radical and the diferric cluster are not near
the active site of the protein that binds
the nucleotide (Figure 16.27). Rather, the
CHAPTER 16
MAGNETIC RESONANCE
369
His His
Inactive
state
Active
state
Active
state
T i m
e
2 e Ϫ
2e
Ϫ
H i s T y r
His Met
H i s N H
3
His Met
H i s
Figure 16.25
A model of the
different states of
cytochrome cd 1 .
Modified from
Allen et al. (2000).
0
3340
3370
Tyrosyl radical
3400
EPR signal
Magnetic field (Gauss)
Figure 16.26 The EPR spectrum of ribonucleotide
reductase. From Stubbe and van der Dork (1998).
9781405124362_4_016.qxd 4/29/08 10:45 Page 369
coordination. As the protein becomes oxidized
with time, large changes are evident in the EPR
spectra. These spectra evolve with primary
features of g values at 2.51, 2.20, and 1.86 that
are consistent with a His–Fe–Tyr coordination.
Also present as a minor component is a highspin species with g values of 6.88 and 5.07.
The combination of these spectroscopic studies
leads to a model of the protein as having three
states (Figure 16.25). Two of these states are
active states: the initial protein conformation that binds the hydroxylamine and the
resulting oxidized state. The third state is an
inactive state that is stable for a prolonged time
but can be returned to the active state by other
regulated proteins.
RESEARCH DIRECTION: RIBONUCLEOTIDE REDUCTASE
A critical step of DNA biosynthesis is the conversion of nucleotides into
deoxynucleotides, which is catalyzed by several classes of enzyme known
collectively as ribonucleotide reductase. Many of these enzymes, class I
ribonucleotide reductases, possess a characteristic EPR spectrum centered
around g = 2.00 with a pronounced hyperfine interaction (Figure 16.26).
Although these enzymes contain two iron atoms that form a diferric
cluster, the EPR spectrum does not arise from the metal cofactors. Rather,
the spectrum arises from a third non-metal
cofactor, a tyrosine radical.
The existence of amino acid radicals was
first discovered in proteins in the 1970s and
they have now been identified in a number of different enzymes (Stubbe & van der
Dork 1998; Stubbe 2003). In each case, the
amino acid radical can be identified primarily by the EPR spectrum, which should
be centered near g = 2.00 and have hyperfine structure. In some cases, the radicals
are transient species, but in the case of
ribonucleotide reductase the tyrosine radical is stable. Surprisingly, the tyrosine
radical and the diferric cluster are not near
the active site of the protein that binds
the nucleotide (Figure 16.27). Rather, the
CHAPTER 16
MAGNETIC RESONANCE
369
His His
Inactive
state
Active
state
Active
state
T i m
e
2 e Ϫ
2e
Ϫ
H i s T y r
His Met
H i s N H
3
His Met
H i s
Figure 16.25
A model of the
different states of
cytochrome cd 1 .
Modified from
Allen et al. (2000).
0
3340
3370
Tyrosyl radical
3400
EPR signal
Magnetic field (Gauss)
Figure 16.26 The EPR spectrum of ribonucleotide
reductase. From Stubbe and van der Dork (1998).
9781405124362_4_016.qxd 4/29/08 10:45 Page 369