Electron paramagnetic resonance evidence for a novel interconversion of [3Fe-4S](+) and [4Fe-4S](+) clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro.
We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis. The activase (nrdG gene) together with S-adenosyl-L-methionine (AdoMet) give rise to a glycyl radical in the NrdD component. A semi-reduced [4Fe-4S](+) cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase. Air exposure of the reduced enzyme gave a [3Fe-4S](+) cluster. The Fe(3)S(4) cluster was convertible to the EPR-active [4Fe-4S](+) cluster by renewed treatment with reducing agents, demonstrating a reversible [3Fe-4S](+)- to-[4Fe-4S](+) cluster conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced [4Fe-4S](+) cluster. Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive. Both reduced states gave the [3Fe-4S](+) EPR signal after air exposure. The iron-sulfur cluster interconversion was also studied in the presence of AdoMet. The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the [3Fe-4S](+) cluster after air exposure was unchanged. The results indicate that an AdoMet-mediated [4Fe-4S](+) center is the native active species that induces the formation of a glycyl radical in the NrdD component.
Liu
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Department of Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden.
Gräslund
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D001426:Bacterial Proteins; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D013294:Lactococcus lactis / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry*; D013997:Time Factors
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D013440:Sulfides; C408542:nrdG protein, Lactococcus lactis; D012436:S-Adenosylmethionine; D007501:Iron; D012264:Ribonucleotide Reductases