BioPHYSICAL chemistry

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 gvalues 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 high-
spin species with gvalues 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 conforma-
tion 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 asribonucleotide 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 num-
ber of different enzymes (Stubbe & van der
Dork 1998; Stubbe 2003). In each case, the
amino acid radical can be identified prim-
arily by the EPR spectrum, which should
be centered near g=2.00 and have hyper-
fine structure. In some cases, the radicals
are transient species, but in the case of
ribonucleotide reductase the tyrosine rad-
ical 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

Time

2e



2e

His Tyr

His Met

His NH

3

His Met

His

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 16.26The EPR spectrum of ribonucleotide

reductase. From Stubbe and van der Dork (1998).