EXAFS CHARACTERIZATION OF THE INTERMEDIATE-X GENERATED DURING THE ASSEMBLY OF THE ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE R2 DIFERRIC TYROSYL RADICAL COFACTOR
Pj. Riggsgelasco et al., EXAFS CHARACTERIZATION OF THE INTERMEDIATE-X GENERATED DURING THE ASSEMBLY OF THE ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE R2 DIFERRIC TYROSYL RADICAL COFACTOR, Journal of the American Chemical Society, 120(5), 1998, pp. 849-860
The assembly of the essential diferric cluster/tyrosyl radical cofacto
r of the R2 subunit of Escherichia coli ribonucleotide reductase from
apoR2 with Fe2+ and O-2 or diferrous R2 with O-2 has been studied by a
variety of rapid kinetic methods. An intermediate X, formally an Fe3/Fe4+ diiron cluster, oxidizes tyrosine 122 to the tyrosyl radical con
comitant with its own reduction to the diferric cluster generating the
R2 cofactor. To characterize the properties of X, rapid freeze quench
methods have been used in conjunction with Mossbauer, ENDOR, and EPR
spectroscopies. These studies are extended here to include rapid freez
e quench EXAFS. A short, 2.5 Angstrom Fe-Fe vector and a 1.8 Angstrom
Fe-O interaction have been identified in nine independent samples of X
. These samples have been generated using both wild-type and a mutant
protein in which the essential tyrosine has been replaced by phenylala
nine (Y122F). The short Fe-Fe interaction is neither present in diferr
ous or diferric R2 nor in samples of X that have aged to decay the int
ermediate. Several structural models which are consistent with the dat
a are presented.