Аннотация
The R2 protein of ribonucleotide reductase contains at the side chain
of tyrosine 122 a stable free radical, which is essential for enzyme
catalysis. The tyrosyl radical is buried in the protein matrix close
to a dinuclear iron center and a cluster of three hydrophobic residues
(Phe-208, Phe-212, and Ile-234) conserved throughout the R2 family.
A key step in the generation of the tyrosyl radical is the activation
of molecular oxygen at the iron center. It has been suggested that
the hydrophobic cluster provides an inert binding pocket for molecular
oxygen bound to the iron center and that it may play a role in directing
the oxidative power of a highly reactive intermediate toward tyrosine
122. We have tested these hypotheses by constructing the following
mutant R2 proteins:F208Y, F212Y, F212W, and I234N. The resulting
mutant proteins all have the ability to form a tyrosine radical,
which indicates that binding of molecular oxygen can occur. In the
case of F208Y, the yield of tyrosyl radical is substantially lower
than in the wild-type case. A competing reaction resulting in hydroxylation
of Tyr-208 implies that the phenylalanine at position 208 may influence
the choice of target for electron abstraction. The most prominent
result is that all mutant proteins show impaired radical half-life;
in three of the four mutants, the half-lives are several orders
of magnitude shorter than that of the wild-type radical. This suggests
that the major role of the hydrophobic pocket is to stabilize the
tyrosyl radical. This hypothesis is corroborated by comparative
studies of the environment of other naturally occurring tyrosyl
radicals.
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