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.

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Journal Article | Research Support, N.I.H., Extramural

Authors

Zhang Y, An X, Stubbe J, Huang M

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