Zhang Y, Liu L, Wu X, An X, Stubbe J, Huang M

The β2 subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (FeIII2-Y·) that is essential for nucleotide reduction. The β2 subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β'). Although only β is capable of iron binding and Y· formation, cells lacking ...

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β' are either dead or exhibit extremely low Y· levels and RNR activity depending on genetic backgrounds. Here we present evidence supporting the model that β' is required for Fe loading and Y· formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in Fe loading into nascent β and are hypersensitive to Fe depletion and the Y·-reducing agent hydroxyurea. Transient induction of β' in a GalRNR4 strain leads to concomitant increase in Fe loading and Y· levels in β. Y· can also be rapidly generated using endogenous iron when permeabilized rnr4 spheroplasts are supplemented with recombinant β', and is inhibited by adding an Fe chelator prior to, but not after, β' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to decrease in both Y· levels and ββ' activity. This result, in combination with previous findings that low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric-Y· cofactor in RNR.

Date: Sep. 20, 2011

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