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.
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.
Lu
Shen
S
Department of Environmental & Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, OR 97006-8921, USA.
Libby
Eduardo
E
Saleh
Lana
L
Xing
Gang
G
Bollinger
J Martin
JM
Moënne-Loccoz
Pierre
P
D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005293:Ferritins / Q000737:chemistry*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009569:Nitric Oxide / Q000737:chemistry*; D010100:Oxygen / Q000494:pharmacology*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis; D013696:Temperature
D021122:Protein Subunits; D009569:Nitric Oxide; D005293:Ferritins; D012264:Ribonucleotide Reductases; D010100:Oxygen