PMID:15311337
| Citation |
Lu, S, Libby, E, Saleh, L, Xing, G, Bollinger, JM Jr and Moënne-Loccoz, P (2004) Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants; implications for the O2 activation mechanism. J. Biol. Inorg. Chem. 9:818-27 |
|---|---|
| Abstract |
The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O(2) to produce a tyrosine radical (Y122.). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [Fe(III)-Fe(IV)] state called compound X, but in related diiron proteins such as methane monooxygenase, Delta(9)-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate (mu-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O(2) activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [(FeNO)(7)](2) center. In the wt and variant proteins, we also observe that both irons bind one NO to form a (FeNO)(7) dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, nu(Fe-NO), delta(Fe-N-O), and nu(N-O) bands are observed at 445, 434 and 1742 cm(-1), respectively, while in the variant proteins the nu(Fe-NO) and delta(Fe-N-O) bands are observed approximately 10 cm(-1) higher and the nu(N-O) approximately 10 cm(-1) lower at 1735 cm(-1). These results demonstrate that all three proteins accommodate fully symmetric [(FeNO)(7)](2) species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O(2) activation process in R2-wt. |
| Links |
PubMed Online version:10.1007/s00775-004-0582-8 |
| Keywords |
Enzyme Activation/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Ferritins/chemistry; Molecular Structure; Mutagenesis, Site-Directed/genetics; Nitric Oxide/chemistry; Oxygen/pharmacology; Protein Subunits/chemistry; Protein Subunits/genetics; Protein Subunits/metabolism; Ribonucleotide Reductases/chemistry; Ribonucleotide Reductases/genetics; Ribonucleotide Reductases/metabolism; Spectrum Analysis; Temperature |
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