Journal
BIOCHEMISTRY
Volume 42, Issue 45, Pages 13269-13279Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi035198p
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- NCRR NIH HHS [1 P20 RR16461-01] Funding Source: Medline
- NIGMS NIH HHS [GM55365, GM58778] Funding Source: Medline
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The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulatin a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mossbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 Angstrom was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 Angstrom Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 Angstrom) than does a sample of anaerobically prepared diferrous R2 (3.4 Angstrom). Possible explanations for this difference in detected Fe-Fe distance include an O-2-induced conformational change prior to covalent chemistry or differing 02 reactivity among multiple diiron(II) forms of the cluster.
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