Abstract
The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2(III)-Y(•)) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr(356) in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ'), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr(376) nor β'-Tyr(323) (Tyr(356) equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ' and an in vivo viability assay show that β-Tyr(376) is essential for RT, whereas Tyr(323) in β' is not. Although the C-terminal tail of β' is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β' C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β' interface.
Highlights
S. cerevisiae ribonucleotide reductase (RNR) comprises ␣ and Ј subunits in an (␣2)m(Ј)n active holoenzyme
We demonstrate that -Tyr376 is not on an essential electron transfer pathway to deliver the reducing equivalent to generate the Fe2III-Y1⁄7 cofactor from the Fe2II or met (Fe2III tyrosyl radical reduced) state either in vitro or in vivo
The C-terminal Tail of Ј Is Dispensable for Fe2III-Y1⁄7 Formation in —We have previously proposed that Ј might use the aspartate and glutamate residues within its C-terminal tail (Figs. 1B and Fig. 2) to bind and deliver FeII to  in the Ј heterodimer in a manner similar to that of copper loading of Sod1 by the chaperone protein Ccs1 [38, 39]
Summary
S. cerevisiae ribonucleotide reductase (RNR) comprises ␣ and Ј subunits in an (␣2)m(Ј)n active holoenzyme. The C termini (30 –35 amino acids) of all 2 structures are disordered [19], and the molecular details of the tail with respect to both itself and ␣ remain unknown Within this C-terminal tail resides a conserved tyrosine residue, Tyr356 in E. coli 2, that has been proposed to play an important role in electron transfer in Fe2III-Y1⁄7 cluster assembly from both the Fe2II and the Fe2III (met) state in 2 [1] and in the long range RT to initiate nucleotide reduction in ␣2 The Ј mutant lacking the C-terminal 8 amino acids (Ј-⌬8aa) is capable of heterodimer formation with  and supporting Fe2III-Y1⁄7 cofactor assembly in , the resulting Ј is catalytically inactive (Ͻ 0.5% WT activity), suggesting an essential role of the Ј tail for interactions with ␣2 to form an active holo-enzyme. Our results for the first time provide in vivo evidence for the predisposition of the C-terminal tails of  and Ј relative to ␣ in the active RNR; these tails interact only with the ␣ in the ␣/ (␣/Ј) pair in the holo-RNR and do not cross over to interact with the adjacent ␣ (see Fig. 2, right panel)
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