Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli.
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 accumulating 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 Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A 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 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.
Baldwin
Jeffrey
J
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Krebs
Carsten
C
Saleh
Lana
L
Stelling
Mindy
M
Huynh
Boi Hanh
BH
Bollinger
J Martin
JM
Riggs-Gelasco
Pamela
P
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015204:Spectroscopy, Mossbauer / Q000379:methods; D013057:Spectrum Analysis / Q000379:methods; D014364:Tryptophan / Q000235:genetics; D014965:X-Rays
D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D014364:Tryptophan; D010105:Oxygenases; C021555:methane monooxygenase; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D000409:Alanine; D010100:Oxygen