Crystal structures of oxidized dinuclear manganese centres in Mn-substituted class I ribonucleotide reductase from Escherichia coli: carboxylate shifts with implications for O2 activation and radical generation.
The di-iron carboxylate proteins constitute a diverse class of non-heme iron enzymes performing a multitude of redox reactions. These reactions usually involve high-valent Fe-oxo species and are thought to be controlled by carboxylate shifts. Owing to their short lifetime, the intermediate structures have so far escaped structural characterization by X-ray crystallography. In an attempt to map the carboxylate conformations available to the protein during different redox states and different ligand environments, we have studied metal-substituted forms of the R2 protein of ribonucleotide reductase from Escherichia coli. In the present work we have solved the crystal structures of Mn-substituted R2 oxidized in two different ways. Oxidation was performed using either nitric oxide or a combination of hydrogen peroxide and hydroxylamine. The two structures are virtually identical, indicating that the oxidation states are the same, most likely a mixed-valent MnII-MnIII centre. One of the carboxylate ligands (D84) adopts a new, so far unseen, conformation, which could participate in the mechanism for radical generation in R2. E238 adopts a bridging-chelating conformation proposed to be important for proper O2 activation but not previously observed in the wild-type enzyme. Probable catalase activity was also observed during the oxidation with H2O2, indicating mechanistic similarities to the di-Mn catalases.
Högbom
M
M
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden.
Andersson
M E
ME
Nordlund
P
P
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism
D005609:Free Radicals; D008345:Manganese; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D010100:Oxygen