Project: Research project

Project Details


DESCRIPTION: The essential cofactor of the enzyme ribonucleotide reductase
consists of an oxo-bridged diiron(III) cluster and an adjacent tyrosyl
radical. The activities of existing anticancer and antiviral drugs (e.g.
hydroxyurea and thiosemicarbazones) derive from their reductive disassembly
of this cofactor and consequent inhibition of DNA synthesis, suggesting that
an understanding of the reaction by which the cofactor is generated might be
of value in design of new pharmacology. The cofactor assembles
spontaneously in vitro when the apo form (lacking iron and radical) of the
enzyme's R2 subunit is incubated with ferrous ions and O2. The assembly
reaction comprises binding of Fe(II) by apo R2, reductive activation of
dioxygen by the resulting diiron(II) cluster, transfer of an "extra
electron" from a third Fe(II) or another reductant to the assembling
cofactor, and one-electron oxidation of a specific tyrosine residue by an
intermediate species. Previous investigations of cofactor assembly into E.
coli R2 suggested 1) that formation of the oxygen-reactive Fe(II)-R2 complex
is a multistep process in which a protein conformational change is rate
limiting, and 2) that the protein facilitates rapid transfer of the "extra
electron" to the reacting iron cluster prior to or during formation of the
first observable intermediate species. The proposed research will use
kinetic and spectroscopic methods in combination with protein engineering
to: 1) characterize Fe(II) binding by the R2 proteins from E. coli, mouse,
and herpes virus, defining the multiple Fe(II)-R2 complexes that are early
intermediates in the assembly reaction and the kinetic pathways by which
they form and decay; 2) define the mechanisms for the delivery of the "extra
electron" and test the hypothesis that, by facilitating this step, the R2
protein ensures the observed one-electron oxidation chemistry to the
exclusion of possible two-electron alternatives (as occur in related diiron
proteins and in the F208Y mutant of E. coli R2); and 3) determine the
chemical mechanism of the altered assembly reaction that occurs in the
site-directed mutant, R2-F208Y, in which the engineered tyrosine residue 208
is ortho hydroxylated (a two-electron reaction). By providing insight into
how the E. coli R3 protein directs the outcome of the assembly reaction and
a foundation for characterizing cofactor assembly into the mammalian and
viral R2s, the proposed research will enhance our understanding of the
biogenesis of this important drug target.
Effective start/end date1/1/9712/31/97


  • National Institute of General Medical Sciences


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