Activation of class III ribonucleotide reductase from E. coli. The electron transfer from the iron-sulfur center to S-adenosylmethionine.
The anaerobic ribonucleotide reductase (ARR) from E. coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals. ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a [4Fe-4S]-containing activating enzyme (beta) and AdoMet under reducing conditions. Here we show that the EPR-active S = 1/2 reduced [4Fe-4S]+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical. These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet. Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT. However, in that case, 2 equiv of methionine per reduced cluster was formed. This unusual stoichiometry and combined EPR and Mössbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active [3Fe-4S] intermediate clusters.
Padovani
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Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DBMS-CB, CEA/CNRS/Université Joseph Fourier, 17, avenue des Martyrs, 38054 Grenoble Cedex 09, France.
Thomas
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F
Trautwein
A X
AX
Mulliez
E
E
Fontecave
M
M
D000693:Anaerobiosis; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D012436:S-Adenosylmethionine; D012264:Ribonucleotide Reductases; D005998:Glycine